Carbohydrate Metabolism

Prof Omar Al-Attas

Biochemistry Department

College of Science King Saud University

Function of Metabolism

1 Synthesis and degrade biomolecules 2 Obtain energy from the environment3 Convert it into cellrsquos own characteristic

molecule4 Polymerize monomeric precursors into

macro molecules

Strategy of metabolism

The basic strategic Is to for ATP reducing power and building block for biosynthesis

1 ATP is the universal currency of energy2 ATP is generated by the oxidation of fuel

molecules3 NADPH electron donor4 Construction of biomolecules from relative small

building blocks5 Biosynthesis and degradative pathways are

distinct

Carbohydrates

bull Compounds made of carbons hydrogen and oxygen

bull General formula isCnH2nOn

bull Principal source of energy in the organism

bull The central molecule is Glucose

Metabolic is controlled in several ways

1 Allosteric modifiers Enzyme catalyzing non-equilibrium reaction are often allosteric proteins subject to the rapid action of ldquofeed ndashbackrdquo or ldquofeed-forwardrdquo control by allosteric modifiers Also the flow of molecules is determined by the amount and activities of certain enzymes rather than by the amount of substrate available

The first irreversible in a pathway (the committed step) is usually an important control element

Generally for an allosteric enzymerate(v) vs [S] isSigmoidal Sigmoidal instead ofhyperbolic

bullSimple enzyme = hyperbolic at low [S] activity fairly highbullComplex allosteric = sigmoidal activity lowbull at low [S] increases greatly in midrangebullEfficiency is better more responsive to [S]

[S]

vsigmoidal

Conthellip

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Strategy of metabolism

The basic strategic Is to for ATP reducing power and building block for biosynthesis

1 ATP is the universal currency of energy2 ATP is generated by the oxidation of fuel

molecules3 NADPH electron donor4 Construction of biomolecules from relative small

building blocks5 Biosynthesis and degradative pathways are

distinct

Carbohydrates

bull Compounds made of carbons hydrogen and oxygen

bull General formula isCnH2nOn

bull Principal source of energy in the organism

bull The central molecule is Glucose

Metabolic is controlled in several ways

1 Allosteric modifiers Enzyme catalyzing non-equilibrium reaction are often allosteric proteins subject to the rapid action of ldquofeed ndashbackrdquo or ldquofeed-forwardrdquo control by allosteric modifiers Also the flow of molecules is determined by the amount and activities of certain enzymes rather than by the amount of substrate available

The first irreversible in a pathway (the committed step) is usually an important control element

Generally for an allosteric enzymerate(v) vs [S] isSigmoidal Sigmoidal instead ofhyperbolic

bullSimple enzyme = hyperbolic at low [S] activity fairly highbullComplex allosteric = sigmoidal activity lowbull at low [S] increases greatly in midrangebullEfficiency is better more responsive to [S]

[S]

vsigmoidal

Conthellip

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Carbohydrates

bull Compounds made of carbons hydrogen and oxygen

bull General formula isCnH2nOn

bull Principal source of energy in the organism

bull The central molecule is Glucose

Metabolic is controlled in several ways

1 Allosteric modifiers Enzyme catalyzing non-equilibrium reaction are often allosteric proteins subject to the rapid action of ldquofeed ndashbackrdquo or ldquofeed-forwardrdquo control by allosteric modifiers Also the flow of molecules is determined by the amount and activities of certain enzymes rather than by the amount of substrate available

The first irreversible in a pathway (the committed step) is usually an important control element

Generally for an allosteric enzymerate(v) vs [S] isSigmoidal Sigmoidal instead ofhyperbolic

bullSimple enzyme = hyperbolic at low [S] activity fairly highbullComplex allosteric = sigmoidal activity lowbull at low [S] increases greatly in midrangebullEfficiency is better more responsive to [S]

[S]

vsigmoidal

Conthellip

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Metabolic is controlled in several ways

1 Allosteric modifiers Enzyme catalyzing non-equilibrium reaction are often allosteric proteins subject to the rapid action of ldquofeed ndashbackrdquo or ldquofeed-forwardrdquo control by allosteric modifiers Also the flow of molecules is determined by the amount and activities of certain enzymes rather than by the amount of substrate available

The first irreversible in a pathway (the committed step) is usually an important control element

Generally for an allosteric enzymerate(v) vs [S] isSigmoidal Sigmoidal instead ofhyperbolic

bullSimple enzyme = hyperbolic at low [S] activity fairly highbullComplex allosteric = sigmoidal activity lowbull at low [S] increases greatly in midrangebullEfficiency is better more responsive to [S]

[S]

vsigmoidal

Conthellip

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Generally for an allosteric enzymerate(v) vs [S] isSigmoidal Sigmoidal instead ofhyperbolic

bullSimple enzyme = hyperbolic at low [S] activity fairly highbullComplex allosteric = sigmoidal activity lowbull at low [S] increases greatly in midrangebullEfficiency is better more responsive to [S]

[S]

vsigmoidal

Conthellip

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Conthellip

2 Covalent modification some regulatory enzymes are controlled by covalent modification ie phosphorylation and dephophorilation

This is rapid and often mediated through the formation of cAMP which in turn causes the conversion of an inactive enzyme into an active enzyme

Example of phosphorylationthe enzyme phosphorylase aphosphorylase a in glycogenutilizationglycogen = polymer of glucose in muscle amp liverglycogen function store amp release glucose

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Breakdown of glycogen (glucose)n + Pi (glucose)n-1+ glucose-1-Pi

Energy source

Enzyme for removing glucose-1-phosphateis phosphorylase a activated by phosphorylation

Another amp another amp another glu-1-Pi is removed

(Pi = inorganic phosphate)

Part of cAMP cascadePart of cAMP cascade

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Conthellip

3 Enzymes levels- The rates of sythesis and degradation of some regulatory enzymes are subject to hormonal factors

4 Compartmentation- The metabolic pathways of eukaryotic cells are marked affected by the presence of compartmentsGlycolysis Pentose P pathway FA sythesis take place in the cytosol

FA oxidation TCA cycle oxydative decarboxylation in mitochrondria

Gluconeogenesis urea cycle synthesis in both compartment

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Conthellip

5 Metabolic specialization of Organs Regulation in higher eukaryotes is profoundly affected and enhanced by the existence of organs with different metabolic roles

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

6 Mass Action Rationbull Is determined by the rate-controlling steps in a

pathway by measuring how close the reactions are to equilibrium

bull Mass action ration Productssubstrates

(measured in the intact cells)

bull If the reaction is close to equilibrium the mass action ration will be close to the equilibrium constant If however the reaction is rate controlling in the cell it will not approach equilibrium and the mass action ration will be lower than equilibrium constant

Conthellip

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Control of Substrate By Insulin

Insulinbull Hormone produce by the beta cells in the

pancreasbull Stored as proinsulin (inactive form) as small

granulesbull Its release is triggered by increased glucose levels

in the bloodbull Stimulates glucose uptake by tissue by binding to

receptors in the cell membrane Permits glucose to enter cell

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Glycolysis

bull First stage of carbohydrate catabolism

bull Is an anaerobic processbull Simple sugar are broken into

pyruvatebull All organism uses this processOverall it uses 1 molecule of

glucose 2 ADP 2 ATP 2 NAD+ and 2 PO4 and 10 different enzymes

bull The net energy production is about 96000 calorie or 8 ATP

6 carbon stage

Requires energy

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Reaction of Glycolysis

3 carbon stage

Double this since 2 pyruvate are made

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Control of Glucose levels

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Glycogenolysis Breakdown of glycogen

Glucagonbull This hormone is also produced in the pancreas

in an inactive formbull Low glucose levels results in it conversion to

an active form and its releasebull Its entry into the liver cells result in the

conversion of glycogen to glucose with glucose being released to the blood

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Glycogen Synthesis

bull It is known as glycogenesisbull Synthesis from glucose occursbull It is carried out by the enzyme glycogen

synthesisbull Occurs when there is excess of glucose in the

body thus helps in the control of glucose in the blood

bull Is stored in the liverbull Occurs in all the tissue of the body but the major

sites are liver and muscle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Regulation of Glycolysis

bull Glycolysis is a sequence in the cytosol which converts

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

bull The Glycolytic pathway describes the oxidation of glucoseto pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2OThe 3 stages of Glycolysis

Conthellip

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

bull Stage 1 is the investment stage 2 mols of ATP areconsumed for each mol of glucosebull Glucose is converted to fructose-16-bisphosphatebull Glucose is trapped inside the cell and at the same timeconverted to an unstable form that can be readily cleavedinto 3-carbon units

bull In stage 2 fructose-16-bisphosphate is cleaved into 2 3-carbon units of glycerladehyde-3-phosphate

bull Stage 3 is the harvesting stage 4 mols of ATP and 2 molsof NADH are gained from each initial mol of glucose ThisATP is a result of substrate-level phosphorylationbull Glyceraldehyde-3-phosphate is oxidized to pyruvate

The 3 stages of Glycolysis

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

bull Reaction 1 Phosphorylation of glucose to glucose-6 phosphatebull This reaction requires energy and so it is coupled to thehydrolysis of ATP to ADP and Pibull Enzyme hexokinase It has a low Km for glucose thus onceglucose enters the cell it gets phosphorylatedbull This step is irreversible So the glucose gets trapped inside thecell (Glucose transporters transport only free glucose notphosphorylated glucose)bull Reaction 2 Isomerization of glucose-6-phosphate to fructose 6-phosphate The aldose sugar is converted into the keto isoformbull Enzyme phosphoglucomutasebull This is a reversible reaction The fructose-6-phosphate is quicklyconsumed and the forward reaction is favored

Step-wise reactions of glycolysis

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Reaction 3 is another kinase reaction Phosphorylation of thehydroxyl group on C1 forming fructose-16- bisphosphatebull Enzyme phosphofructokinase This allosteric enzyme regulatesthe pace of glycolysisbull Reaction is coupled to the hydrolysis of an ATP to ADP and Pibull This is the second irreversible reaction of the glycolytic pathwaybull Reaction 4 fructose-16-bisphosphate is split into 2 3-carbonmolecules one aldehyde and one ketone dihyroxyacetonephosphate (DHAP) and glyceraldehyde 3-phosphate (GAP)bull The enzyme is aldolasebull Reaction 5 DHAP and GAP are isomers of each other and canreadily inter-convert by the action of the enzyme triose-phosphateisomerasebull GAP is a substrate for the next step in glycolysis so all of theDHAP is eventually depleted So

Conthellip

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

bull Upto this step 2 molecules of ATP were required for eachmolecule of glucose being oxidizedbull The remaining steps release enough energy to shift thebalance sheet to the positive side This part of the glycolyticpathway is called as the payoff or harvest stagebull Since there are 2 GAP molecules generated from eachglucose each of the remaining reactions occur twice for eachglucose molecule being oxidizedbull Reaction 6 GAP is dehydrogenated by the enzymeglyceraldehyde 3-phosphate dehydrogenase (GAPDH) Inthe process NAD+ is reduced to NADH + H+ from NADOxidation is coupled to the phosphorylation of the C1carbon The product is 13-bisphosphoglycerate

Conthellip

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Reaction 7 BPG has a mixed anhydride a high energy bondat C1 This high energy bond is hydrolyzed to a carboxylicacid and the energy released is used to generate ATP fromADP Product 3-phosphoglycerate Enzymephosphoglycerate kinasebull Reaction 8 The phosphate shifts from C3 to C2 to form 2-phosphoglycerate Enzymephosphoglycerate mutasebull Reaction 9 Dehydration catalyzed by enolase (a lyase) Awater molecule is removed to form phosphoenolpyruvatewhich has a double bond between C2 and C3bull Reaction 10 Enolphosphate is a high energy bond It ishydrolyzed to form the enolic form of pyruvate with thesynthesis of ATP The irreversible reaction is catalyzed bythe enzyme pyruvate kinase Enol pyruvate quickly changes to keto pyruvate which is far more stable

Conthellip

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Glycolysis Energy balance sheet

bull Hexokinase - 1 ATPbull Phosphofructokinase -1 ATPbull GAPDH +2 NADHbull Phsophoglycerate kinase +2 ATPbull Pyruvate kinase +2 ATPTotal molecule of glucose +2 ATP +2 NADH

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Fate of Pyruvate

bull NADH is formed from NAD+ during glycolysisbull The redox balance of the cell has to be maintained forfurther cycles of glycolysis to continuebull NAD+ can be regenerated by one of the followingreactions pathwaysbull Pyruvate is converted to lactatebull Pyruvate is converted to ethanolbull In the presence of O2 NAD+ is regenerated by ETCPyruvate is converted to acetyl CoA which entersTCA cycle and gets completely oxidized to CO2

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Lactate Fermentation

Formation of lactate catalyzed by lactate dehydrogenaseCH3-CO-COOH + NADH + H+10487731048773CH3-CHOH-COOH + NAD+bull In highly active muscle there is anaerobic glycolysisbecause the supply of O2 cannot keep up with the demandfor ATPbull Lactate builds up causing a drop in pH which inactivatesglycolytic enzymes End result is energy deprivation andcell death the symptoms being pain and fatigue of themusclebull Lactate is transported to the liver where it can bereconverted to pyruvate by the LDH reverse reaction

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Ethanol fermentation

bull Formation of ethanol catalyzed by 2 enzymesbull Pyruvate decarboxylase catalyzes the first irreversiblereaction to form acetaldehydeCH3-CO-COOH 1048773 CH3-CHO + CO2bull Acetaldehyde is reduced by alcohol dehydogenase is areversible reactionCH3-CHO + NADH + H+ 10487731048773 CH3CH2OH + NAD+bull Ethanol fermentation is used during wine-making

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Conthellip

Fructose is phosphorylated by fructokinase (liver) or hexokinase(adipose) on the 1 or 6 positions respbull Fructose-6-phosphate is an intermediate of glycolysisbull Fructose-1-phosphate is acted upon by an aldolase-like enz thatgives DHAP and glyceraldehydebull DHAP is a glycolysis intermediate and glyceraldehyde can bephosphorylated to glyceraldehyde-3-Pbull Glycerol is phosphorylated to G-3-P which is then converted toglyceraldehyde 3 phosphatebull Galactose has a slightly complicated multi-step pathway forconversion to glucose-1-phosphatebull gal 1048773 gal-1-P 1048773 UDP-gal 1048773 UDP-glc 1048773 glc-1-Pbull If this pathway is disrupted because of defect in one or more enzinvolved in the conversion of gal to glc-1-P then galactoseaccumulates in the blood and the subject suffers from galactosemia which is a genetic disorder an inborn error of metabolism

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

The Glycolytic pathway describes the oxidation of glucose to pyruvate with the generation of ATP and NADHbull It is also called as the Embden-Meyerhof Pathwaybull Glycolysis is a universal pathway present in all organismsfrom yeast to mammalsbull In eukaryotes glycolysis takes place in the cytosolbull Glycolysis is anaerobic it does not require oxygenbull In the presence of O2 pyruvate is further oxidized to CO2In the absence of O2 pyruvate can be fermented to lactate orethanolbull Net Reaction Glucose + 2NAD+ + 2 Pi + 2 ADP =2 pyruvate + 2 ATP + 2 NADH + 2 H2O

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

The citric acid cycle

1 Final stage for the metabolism of carbohydrates fats and amino acids

2 Oxidative cycle3 Requires oxygen4 Aerobic5 Also called the Krebs

cycle for Hans Krebs who first described it

bull Citric acid cyclebull Also known as

tricarboxylic acid cycle (TCA cycle) or the Krebss cycle

bull Pyruvate formed after glycolysis undergoes oxydative decarboxylation to form acytyl coenzyme A

bull The acetyl co A gets completely oxidzed into CO2 by TCA cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Control of Citric acid cycle

bull Several route for controlling the cyclebull Insufficient Oxygenbull Reduced energy demand causes a build up of ATP

NADH inhibits ndashpyruvate conversion to acetyl CoA conversion Acetyl CoA production of citrate (ATP only) Some intermediate steps in the cycle

bull Excess ADP will stimulate many of these steps to things go faster

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

The Citric Acid Cycle

bull In aerobic organisms pyruvate (formed through

glycolysis) oxidized to CO2 and acetyl-CoA using

coenzyme A

bull Subsequent oxidation of acetyl group carried out using

the citric acid cycle

bull Citric acid cycle is amphibolic

ndash Catabolic and anabolic

ndash Aerobic catabolism of carbohydrates lipids and amino

acids merge at citric acid cycle

bull Oxidized acetyl-CoA formed from metabolism of all

three

ndash Intermediates of citric acid cycle are starting points for

many biosynthetic pathways

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

In Mitochondria

In Cytosol

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

The amphibolic Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

conthellip

bull Controlled metabolic oxidation ndash Compounds oxidized in discrete stepsndash Enzymatic transfer of electrons to O2

bull Analogous to combustion of organic compounds to produce CO2 and H2O and heatndash Energy released largely conserved when NAD+ and ubiquinone (Q) are reduced to NADH

and ubiquinol (QH2)ndash Re-oxidation of reduced coenzymes produce more energy (ATP) through electron

transport and oxidative phosphorylation

bull akandash tricarboxylic acid (TCA) cycle (due to tricarboxylate intermediates)ndash Krebs cycle (for biochemist Hans Krebs)

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Net reaction

bull Circular pathwayndash Regenerate oxaloacetate starting material in last step of cyclendash Cycle is a multi-step enzyme can catalyze unlimited acetyl groups

bull Carbon flowndash 2 carbons that enter the cycle are not the same

carbons lost as CO2

bull Series of 8 enzyme-catalyzed reactionsbull Transfer of 4 pairs of electronsbull Production of energy-rich molecules

ndash Most of energy released is conserved in the form of reduced coenzymes NADH and FADH2 (or QH2)

ndash Oxidation of reduced coenzymes through electron transport and production of ATP from ADP and Pi through oxidative phosphorylation

bull 9 ATP can be formed from one cycle when 4 pairs of electrons transferred to O2

ndash Step 5 is substrate-level phosphorylation to produce ATP or GTP depending on type of cell

bull Net reactionAcetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O rarr

2 CO2 + 3 NADH + FADH2 + CoA + GTP + 3 H+

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

The pathway

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Synthesis of acetyl-CoA

H

N

H

CH2

O OH

H HO

N

N

N

NH2

P

O

OO

OPOPOCH2CCH

CHNCH2CH2C

HNCH2CH2HS

O O

OH

CH3

CH3 O

O

O

O

bull Not part of cycle but must occur firstbull First step entry of pyruvate into the mitochondrian

ndash In aerobic cells all enzymes of the citric acid cycle are located within the mitochondrionndash Mitochondrion enclosed by a double membranendash Pyruvate passes through outer membrane via aqueous channels formed by transmembrane

proteins (porins)ndash Pyruvate translocase is a protein embedded in the inner mitochondrial membrane which

transports pyruvate from the intermembrane space to the mitochondrial matrix (interior space of the mitochondrion)

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Conversion of pyruvate to acetyl-CoA

bull Oxidative decarboxylationndash Series of 5 reactionsndash Irreversiblendash Mechanism is highly complicated

bull Catalyzed by complex of enzymes and cofactorsndash Pyruvate dehydrogenase complexndash Multi-enzyme structure located

in mitochondrial matrixndash Contains multiple copies of three

non-covalently associated enzymes

and five coenzymes

ndash E1 and E3 surround core of 24-60 E2 chains

( chains depends on type of cell)

bull Overall reaction CH3C(O)CO2

- + NAD+ + CoASH rarr CH3C(O)-SCoA + NADH + CO2

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

1 Formation of citrate

bull Oxaloacetate reacts with acetyl-CoA to form

citrate and coenzyme A

bull Aldol condensation

ndash Only C-C bond-forming reaction in cycle

bull Irreversible

bull Enzyme = citrate synthase

CO2

C

CH2

CO2

O+

S

C

CH3

CoA

O

H2O

H+

CO2

CH2

C

CH2

CO2

HO CO2 + HS-CoA

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Citrate synthasebull Dimer of two identical subunitsbull Changes in conformation

ndash Binding of oxaloacetatendash Domains move closer to form binding

site for acetyl-CoAndash Formation of intermediatendash Enzyme closes around intermediate

bull Prevent side reactions by shielding thiol ester linkage of acetyl-CoA from hydrolysis by solvent

bull Intermediate hydrolyzed by bound water molecule

ndash Enzyme opens and products leave active site

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

2 Isomerization of citrate to isocitrate

H2O

CO2

CH2

C

CH2

CO2

HO CO2

CO2

CH2

C

CH

CO2

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

H2O

bull Citrate is a 3deg alcoholndash Cannot be oxidized to keto acid

bull Isocitrate is a 2deg alcoholndash Easily oxidized

bull Mechanismndash First step elimination of H2O to from alkene

intermediate (cis-aconitate)ndash Second step stereospecific addition of water to

form (2R 3S)-isocitratendash Reaction near equilibrium

bull Enzyme = aconitasendash aka aconitate hydratasendash Named for intermediatendash Binds C3 carboxylate and hydroxyl groups

bull Substrate positioning essential for stereospecificity

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

3 Oxidative decarboxylation of isocitrate to form -ketoglutarate

CO2

CO2

CH2

HC

CH

CO2

CO2

HO

NAD+

NADH + H+

CO2

CH2

HC

C

CO2

CO2

O

CO2

CH2

CH2

C

CO2

OH+

bull First of four oxidation-reduction reactionsndash NAD+ is oxidizing agent

bull Mechanismndash First step alcohol oxidized by transfer of H- from

C2 to NAD+

bull Intermediate = oxalosuccinate an unstable -keto acid

bull First molecule of NADH formed

ndash Second step intermediate undergoes -decarboxylation to form an -keto acid which is released from enzyme

ndash First molecule of CO2 produced

ndash Irreversiblendash One of rate-limiting steps in cycle

bull Enzyme = isocitrate dehydrogenase

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

4 Oxidative decarboxylation of -ketoglutarate to form succinyl-CoA

NAD+

CO2

CH2

CH2

C

CO2

O

NADH

HS-CoA

CO2

CO2

CH2

CH2

C

S-CoA

O

bull Catalyzed by multi-enzyme -ketoglutarate dehydrogenase complex

ndash -ketoglutarate dehydrogenase (E1)

ndash Dihydrolipoamide succinyltransferase (E2)

ndash Dihyrdolipoamide dehydrogenase (E3)

ndash Analogous to pyruvate-to-acetyl-CoA reaction catalyzed by pyruvate dehydrogenase complex

bull Same coenzymesbull Similar complicated mechanism

bull Product is high-energy thioesterbull Key regulatory step of citric acid cyclebull Second molecule of NADH produced

bull Second molecule of CO2 produced

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Halfway through the cyclehellip

bull So farhellipndash Net oxidation of two carbon atoms to produce two

molecules CO2

bull In the next four reactionshellipndash Four-carbon succinyl group of succinyl CoA converted

back to oxaloacetatendash As oxaloacetate is regenerated additional acetyl- CoA

enters the citric acid cycle to be oxidized

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

5 Conversion of succinyl-CoA to succinate

GDP + Pi

CO2

CH2

CH2

CO2

GTP + HS-CoA

CO2

CH2

CH2

C

S-CoA

O

bull Substrate-level phosphorylationndash Cleavage of high-energy thioester bondndash Free energy conserved through the synthesis of nucleoside

triphosphatebull GTP in mammalsbull ATP in plants and bacteria

ndash GDP regenerated and ATP produced from the reaction of GTP with ADP

bull GTP + ADP GDP + ATPbull Nucleoside diphosphate kinase

bull Enzyme = succinyl-CoA synthetasendash aka succinate thiokinase

bull Mechanismndash Phosphate displace CoA from bound succinyl-CoA moleculendash Phosphoryl group transfers to His residue of enzymendash Succinate releasedndash Phosphoryl group transferred to GDP (or ADP)

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Succinyl-CoA synthetase mechanism

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

6 Oxidation of succinate to fumaratebull Dehydrogenation (loss of H2 oxidation)

ndash Stereospecific to form trans double bond onlybull Catalyzed by succinate dehydrogenase complex

ndash aka succinate dehydrogenasendash aka Complex IIndash Embedded in inner mitochondrial membrane rather than in

mitochondrial matrixbull Oxidation of alkane requires stronger oxidizing agent than NAD+

(hence FAD)ndash FADH2 produced is re-oxidized by

coenzyme ubiquinone (Q) to reform FAD and ubiquinol (QH2)

bull Competitive inhibitor = malonatendash -O2C-CH2-CO2

-

ndash Binds to active site through carboxylate groupsndash Cannot undergo dehydrogenationndash Inhibition reactions used by Krebs to determine

citric acid cycle reaction sequencebull Symmetrical molecule evenly distributes carbons

in remainder of products throughout the cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

7 Hydration of fumarate to form L-malate

H2O

CO2

C

C

O2C

H

H

C

CH2

CO2

CO2

HHO

bull Reversible reaction near equilibrium

bull Stereospecificity1 trans addition of water to double

bond of fumarate

2 Only trans double bond will react

bull Enzyme = fumarasendash aka fumarate hydratase

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

8 Oxidation of L-malate to regenerate oxaloacetate

NAD+

NADH + H+

C

CH2

CO2

CO2

HHO

C

CH2

CO2

CO2

O

bull Formation of third molecule of NADH

bull Reaction is endergonic and concentration of

product is low at equilibrium

ndash Next reaction in cycle (1) is highly exergonic

ndash Product used immediately

bull Enzyme = malate dehydrogenase

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle

Regulation of the Citric Acid Cycle

bull Achieved by the modulation of key enzymes and the availability of certain substrates

bull Recall that build-up of citrate slows glycolysisproduction of pyruvatebull Regulation also depends on continuous supply of acetyl-CoA (from pyruvate)

NAD+ FAD and ADPbull Regulated enzymes in the citric acid cycle

ndash Citrate synthase (reaction 1)bull Allosterically inhibited by high concentrations

of citrate succinyl-CoA NADH ATP

ndash Isocitrate dehydrogenase (reaction 3)bull Activity stimulated by ADP NAD+ and Ca2+ (muscle)bull Inhibited by ATP and NADH

ndash -Ketoglutarate dehydrogenase (reaction 4)bull Inhibited by ATP GTP NADH and succinyl-CoA

ndash All three of these enzymes catalyze reactions that

represent important metabolic branch points

• PowerPoint Presentation
• Function of Metabolism
• Strategy of metabolism
• Carbohydrates
• Metabolic is controlled in several ways
• Slide 6
• Conthellip
• Slide 8
• Slide 9
• Slide 10
• 6 Mass Action Ration
• Control of Substrate By Insulin
• Glycolysis
• Reaction of Glycolysis
• Control of Glucose levels
• Glycogenolysis Breakdown of glycogen
• Glycogen Synthesis
• Regulation of Glycolysis
• Slide 19
• The 3 stages of Glycolysis
• Step-wise reactions of glycolysis
• Slide 22
• Slide 23
• Slide 24
• Glycolysis Energy balance sheet
• Fate of Pyruvate
• Lactate Fermentation
• Ethanol fermentation
• Slide 29
• Slide 30
• The citric acid cycle
• Control of Citric acid cycle
• The Citric Acid Cycle
• Slide 34
• The amphibolic Citric Acid Cycle
• conthellip
• Net reaction
• The pathway
• Synthesis of acetyl-CoA
• Conversion of pyruvate to acetyl-CoA
• 1 Formation of citrate
• Citrate synthase
• 2 Isomerization of citrate to isocitrate
• 3 Oxidative decarboxylation of isocitrate to form a-ketoglutarate
• 4 Oxidative decarboxylation of a-ketoglutarate to form succinyl-CoA
• Halfway through the cyclehellip
• 5 Conversion of succinyl-CoA to succinate
• Succinyl-CoA synthetase mechanism
• 6 Oxidation of succinate to fumarate
• 7 Hydration of fumarate to form L-malate
• 8 Oxidation of L-malate to regenerate oxaloacetate
• Regulation of the Citric Acid Cycle