crystal.res.ku.edu taksnotes biol 638 notes chp 16

8/20/2019 Crystal.res.Ku.edu Taksnotes Biol 638 Notes Chp 16

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Chapter-16 Takusagawa’s Note! 1

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Chapter 16: Citric Acid Cycle

1. CITRIC ACID CYCLE OVERVIEW (all carried out in mitochondrial matrix) - Citric acid cycle is also called Krebs cycle or tricarboxylic acid (TCA) cycle.- Citric acid cycle is the process that the pyruvate produced in glycolysis is further oxidized to

3CO2 to produce 4NADH, FADH2 and GTP (ATP). The NADH and FADH2 are utilized to produce the “energy currency” ATP in oxidative phosphorylation.

H3C C COO-

O

H3C C S

O

CoA

CoASH + NAD+

CO2 + NADH

COO-

C

CH2

COO-

O

COO-

CH2

C COO-

HO

CH2

COO-

COO-

CH2

C COO-

CH

COO-

COO-

CH2

C COO-

H

C

COO-HHO

COO-

CH2

C COO-

HC

COO-O

COO-

CH2

CH2

C

COO-O

COO-

CH2CH2

C

S

O

CoA

COO-

CH2

CH2

COO-

COO-

CH

HC

COO-

COO-

CHHO

COO-

CH2

H2O

CoA

H2O

H2O

NAD+

NADH

+ H+

CO2

CoASHCO2

NAD+NADH

+ H+

CoASH

GDP + Pi

GTP

FAD

FADH2

H2O

NAD+

NADH + H+

Citrate

cis-Aconitate

1. citrate synthase

2. aconitase

Isocitrate

3. isocitrate dehydrogenase

Oxalosuccinate

α-Ketoglutarate

4. α-ketoglutarate

dehydrogenase

Succinyl-CoA

5. succinyl-CoA synthetase

Succinate

6. Succinate dehydrogenase

7. fumarase

Fumarate

L-malate

8. malate dehydrogenase

1

2

2

3

34

5

6

7

8

Oxaloacetate

Acetyl-CoA

pyruvatedehydrogenase

Pyruvate

• 2CO2 are not come from

the original acetate.


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- Pyruvate generated from glycolysis is converted to acetyl-CoA before entering the citric acidcycle.

- At the initial reaction, acetyl group from acetyl-CoA and oxaloacetate react to form citrate.- 3NADH, FADH2 and GTP are generated from one acetyl-CoA oxidation.- 2CO2 are released from the portion of oxaloacetate.

- At the final reaction, oxaloacetate is regenerated.- Overall reaction in the citric acid cycle is:3NAD+ + FAD + GDP + Pi + acetyl-CoA → 3NADH + FADH2 + GTP + CoA + 2CO2

From glucose:Glucose + 2NAD+ + 2ADP + 2Pi → 2pyruvate + 2NADH + 2ATP2pyruvate + 2NAD+ + 2CoA → 2acetyl-CoA + 2NADH + 2CO2 2acetyl-CoA + 6NAD+ + 2FAD + 2GDP + 2Pi → 6NADH + 2FADH2 + 2GTP + 2CoA + 4CO2 2GTP + 2ADP → 2ATP + 2GDP .Glucose + 10NAD+ + 4ADP + 4Pi + 2FAD → 10NADH + 2FADH2 + 4ATP + 6CO2

→ 30ATP + 4ATP + 4ATP = 38ATP

2. METABOLIC SOURCES OF ACETYL-COENZYME A

- Pyruvate is converted to acetyl-CoA before entering the citric acid cycle.- The function of coenzyme A is a carrier of acetyl and other acyl group.- Acetyl-CoA is a “high-energy” compound since it has a “high energy” S~C bond which

releases ∆G°’ = -31.5 kJ/mol by hydrolysis.

Acetyl-coenzyme A (acetyl-CoA)

S

CH2CH2

NH

C

CH2

CH2

NH

C

CC

H2C

C CH3

O

O P O P O CH2

O

O

HHOCH3H3C O

O-

O

O-

O

O OH

P

O

O--O

N

N N

N

NH2

Acetyl group

β-mercaptoethyl-amine residue

Pantothenicacid residue

Adenosine-3'-phosphate

High energy bond


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A. Pyruvate dehydrogenase is a mul tienzyme complex - Acetyl-CoA is formed from pyruvate through oxidative decarboxylation by a multienzyme

complex named pyruvate dehydrogenase.Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH

- Pyruvate dehydrogenase multienzyme complex consists of:

1. Pyruvate dehydrogenase (E1)2. Dihydrolipoyl transacetylase (E2)3. Dihydrolipoyl dehydrogenase (E3)

- Mul tienzyme complexes have catalytic advantages :1. Rates of a series of reactions are enhanced since short diffusion distance.2. Side reactions are minimized.3. Reactions may be coordinately controlled.

- The following coenzymes and prosthetic groups are required in pyruvate dehydrogenasemultienzyme complex:

- Thiamine pyrophosphate (TPP, Fig. 16-27) decarboxylase " - Flavin adenine dinucleotide (FAD, Fig. 14-28) redox # See next page.- Nicotinamide adenine dinucleotide (NAD+, Fig. 12-2) redox $ - Coenzyme A (see previous page) acetyl-carrier- Lipoamide (prosthetic group) acetyl-carrier


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Acetyl-CoA formation occurs in fi ve reactions

1. Pyruvate dehydrogenase (E1), a TPP-requiring enzyme, decarboxylates a pyruvate with theintermediate formation of hydroxyethyl-TPP. This is the same reaction catalyzed by yeast pyruvate decarboxylase (pyruvate→ acetylaldehyde + CO2).


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2. Hydroxyethyl group is transferred to the next enzyme (dihydrolipoyl transacetylase (E2)).The hydroxyethyl group carbanion attacks the lipoamide disulfide of E2 and eliminate theTPP to form acetyl-dihydrolipoamide-E2.

H+

B:

3. The acetyl group is transferred to CoA to yield acetyl-CoA and dihydrolipoamide-E2.

4. Dihydrolipoamide-E2 is oxidized by dihydrolipoyl dehydrogenase (E3)

5. Reduced E3 is reoxidized by NAD+. Initially the enzyme’s sulfhydryl groups (-SH) are

reoxidized by the enzyme-bound FAD, yielding FADH2, then FADH2 is reoxidized by NAD+, producing NADH.

+ +


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The li poyll ysyl arm transfers intermediates between enzyme subuni ts - Lipoyllysyl arm is quite long (14 Å).

HNNH

OO

S S

14 Å

Lipollysyl arm(fully extended)

Arsenic compounds are poisonous because they covalently bind to the vicinal (adjacent) dithiolsof dihydrolipoamide.


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B. Control of pyruvate dehydrogenase Product inhibition - When the relative concentrations of NADH and acetyl-CoA are high, the reversible reactions

catalyzed by E2 and E3 are driven backwards. Therefore formation of acetyl-CoA isinhibited.

- Thus the E2 and E3 activities are controlled by product inhibition (acetyl-CoA for E2 and NADH for E3).

Covalent modifi cation (Eukaryotic complex only)- E1 is regulated by phosphorylation/dephosphorylation. When the Ser of E1 is

phosphorylated, the enzyme is inactivated.

Activators of phosphatase: Mg2+, Ca2+ Activators of kinase: Acetyl-CoA, NADH

Inhibitors of kinase: Pyruvate, ADP, Ca2+, high Mg2+, K +

- Remember: Insulin inhibits phosphorylation and activates dephosphorylation in order toreduce the [glucose] in blood at the starting point of glycolysis.

- Now, insulin also works to reduce the end product of glycolysis, i.e., activatesdephosphorylation of E1 to convert pyruvate to acetyl-CoA.

- Acetyl-CoA is not only the fuel of citric acid cycle, but also the precursor of fatty acids.

Insulin activates


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3. Enzymes of the citric acid cycle A. Ci trate synthase

- catalyzes the condensation of acetyl-CoA and oxaloacetate.

CoA-SHH2O

+ HO C COO-

H2CCOO

-

H2C C O

-

O

O C COO-

H2CCOO

-

H3C CO

S CoA

Acetyl-CoA Oxaloacetate Citrate

∆G°’ = -32.2 kJ/mol

Reaction mechani sm

1. Asp-375 acts as a base to remove a proton from the methyl group of acetyl-CoA. His-274acts as an acid to protonate the enolate oxygen.

2. Citryl-CoA is formed in a second concerted acid-base catalysis. His-320 acts as acid, andHis-274 acts as base.

3. Citryl-CoA is hydrolyzed to citrate and CoA. This hydrolysis (∆G°’ = -31.5 kJ/mol) pullsthe reaction 1 and 2.

H2OCoASH

1

3


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B. Aconitase

- catalyzes the reversible isomerization of citrate and isocitrate.

HO C COO-

H2C COO-

C COO-

H

H

H2O

C COO-

H2C COO-

C COO-

H

H C COO-

H2C COO-

C COO-

H

HO

H2O

Citrate cis-Aconitate Isocitrate ∆G°’ = 13.3 kJ/mol

Reaction mechanism - Aconitase contains a covalently bound [4Fe-4S] iron-sulfur cluster, which is required for

catalytic activity. The Fea is coordinated by the hydroxyl and the central carboxyl groups.1. His-101 acts as an acid to eliminate -OH as water, and Ser-642 acts as a base to eliminate a

proton from C2.2. cis-Aconitate intermediate is flipped by 180° so that C2 and C3 are exchanged their

positions.3. The reversed acid-base catalysis is taken place to yield (2R,3S)-isocitrate.


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F luorocitrate inh ibits aconitase

- Fluoroacetate, one of the most toxic small molecules (LD50 = 0.2 mg/kg), is converted to(2R,3R)-fluorocitrate, which specifically inhibits aconitase since Ser-642 cannot remove the proton at C2.

C. NAD + -dependent isocitr ate dehydrogenase

- catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate to produce CO2 and NADH.

H C COO-

H2C COO-

C COO-

H

HO

Isocitrate

CH2

H2C COO-

C COO-

O

NAD+ NADH + H

+

CO2+

α-Ketoglutarate ∆G°’ = -20.9 kJ/mol

- There are two isozymes in mammalian cells.1. NAD+-dependent form is in mitochondria and requires an Mn2+ or Mg2+.2. NADP+-dependent form is in both cytosol and mitochondria.

D. α αα α -Ketoglutarate dehydrogenase

- catalyzes the oxidative decarboxylation of an α-keto acid, releasing CO2, forming succinyl-CoA and reducing NAD+ to NADH.

+ CO2CH2

H2C COO-

C

O

S-CoA

Succinyl-CoA

NADHNAD+CoA-SH

α-Ketoglutarate

CH2

H2C COO-

C COO-

O

∆G°’ = -33.5 kJ/mol

Less acidic

Less toxic Very toxic


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- α-Ketoglutarate dehydrogenase is a multienzyme complex that consists of α-ketoglutaratedehydrogenase (E1), dihydrolipoyl transsuccinylase (E2), and dihydrolipoyl dehydrogenase(E3).

- The overall reaction closely resembles that are catalyzed by the pyruvate dehydrogenasemultienzyme complex, i.e.,

1. Decarboxylation -----------------------E1 2. Succinyl group transfer " E2 3. Succinyl-CoA formation. $ 4. Oxidation of E2. " E3 5. Reduction of NAD+. $

E. Succinyl-CoA synthetase

- hydrolyzes the “high-energy” compound succinyl-CoA with the coupled synthesis of a “high-energy” nucleosidetriphosphate (GTP).

Succinate

COO-

CH2

CH2

COO-

CoA-SHCH2

H2C COO-

C

O

S-CoA

Succinyl-CoA

GTPGDP + Pi

∆G°’ = -2.9 kJ/mol

- The succinyl~CoA thioester bond energy is preserved through the formation of a series of“high-energy” phosphate (~Pi). The succinate formation is as follows:

Succinyl~CoA

Pi

CoASH

Succinyl~Pi

E-His~Pi

GDPE-His

GDP~Pi (GTP)1

23 E-His

Succinate

- GTP is converted to ATP by nucleoside diphosphate kinase.GTP + ADP ↔ GDP + ATP ∆G°’ = 0 kJ/mol


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F . Succinate dehydrogenase - catalyzes stereospecific dehydrogenation of succinate to fumarate and produces FADH2.

FumarateSuccinate

COO-

C H

CH

COO-

H

H

COO-

C H

CH

COO-

FAD FADH2

∆G°’ = 0 kJ/mol

- The FAD in succinate dehydrogenase is covalently bound to the enzyme. Thus, FADH2 cannot be oxidized as a cofactor. FADH2 is oxidized by the electron transport chain reaction(See Chapter-17).

- For the reason, succinate dehydrogenase is the only membrane-bound citric acid cycleenzyme. The others are dissolved in the mitochondrial matrix.

- The enzyme is strongly inhibited by malonate (structural analog of succinate).

COO-

C H

CH

COO-

H

HCOO

-

C H

COO-

H

Malonate Succinate In general, FAD and NAD+ are involved in different oxidation-reduction reactions.- For example,

C H

CH

FAD FADH2C H

H C

H

H

Alkane Alkene - The oxidation of alkane to alkene produces ∆G°’ ≈ -42 kJ/mol, whereas the FAD to FADH2

reduction requires ~42 kJ/mol (FAD + 2H+ + 2e- → FADH2, ∆E°’ = -0.219 V = (∆G°’ = 42kJ/mol)). Thus, the oxidation of alkane to alkene is just enough to reduce FAD to FADH2, but not enough to reduce NAD+ to NADH + H+ (∆G°’ = 61 kJ/mol).

- The oxidation of alcohol to aldehyde (or ketone) produces more energy than the above case.

Aldehyde or ketoneAlcohol

NAD+

NADH + H+

C

C

HH

O

C

C

HH

OHH

- Alcohol → aldehyde (or ketone) ∆G°’ ≈ -61 kJ/mol

NAD+ + 2H+ + 2e- → NADH + H+ ∆E°’ = -0.315 V (∆G°’ = 61 kJ/mol)- The oxidation of alcohol to aldehyde is sufficient to reduce NAD+ to NADH2.


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G. Fumarase - catalyzes the hydration of fumarate’s double bond to form L-malate.

L-Malate

H2O COO-

C H

H

COO-

H C

HO

Fumarate

COO-

C H

CHCOO

-

∆G°’ = -3.8 kJ/mol

H. Malate dehydrogenase

- catalyzes the oxidation of L-malate’s hydroxyl group to ketone in a NAD+-dependentreaction, regenerating oxaloacetate.

NADH + H+

NAD+COO

-

C H

H

COO-

H C

HO

L-Malate Oxaloacetate

COO-

C O

H

COO-

H C

∆G°’ = 29.7 kJ/mol

- This reaction is relatively high endergonic reaction (∆G > 0).- However, the following two reasons, this reaction occurs.

1. [Oxaloacetate] is very low at equilibrium, i.e., RTln K eq becomes negative where

K eq =[ ][ ]

[ ][ ]oxaloacetate NADH

malate NAD+ < 1, i.e., ln K eq < 0.

2. The subsequent reaction (formation of citrate from oxaloacetate and acetyl-CoA) that ishighly exergonic pulls this reaction since the hydrolysis of “high-energy” thioester bond

of acetyl-CoA releases ∆G°’ = -31.5 kJ/mol energy. This is a reason why acetyl-CoAenters the citric acid cycle.


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I . I ntegration of the citri c acid cycle - Citric acid cycle results in the following chemical transformations.

1. One acetyl group (-COCH3) → 2CO2 (4-electron pair process).

CoA S C CH3

O

3H2O+ 2CO2 + CoA SH + 8H+ + 8e

-

2. Reduction of three NAD+ to three NADH (3-electron pairs process) and equivalent to

9ATP generation, i.e., 3NAD+ + 6H+ + 6e- → 3NADH + 3H+ 3. Reduction of one FAD to FADH2 (1-electron pairs process) and equivalent to 2ATP

generation, i.e., FAD + 2H+ + 2e- → FADH2 4. Generation of one GTP (ATP).

- Four electron pairs generated by one acetyl group oxidation are carried by 3NADH andFADH2 to the oxidative phosphorylation pathway to generate 11ATP.

- Thus, citric acid cycle generates 12ATP from one acetyl group and sends 4-electron pairs (8electrons) to electron-transport chain, where they reduce two molecules of O2 to 4H2O, i.e.,

2O2 + 8H+ + 8e- → 4H2O.


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4. REGULATION OF THE CITRIC ACID CYCLE - Citrate synthase, isocitrate dehydrogenase and αααα-ketoglutarate dehydrogenase are the

citric acid cycle’s rate-controlling enzymes because those ∆G are negative.- The citric acid cycle reactions are carried out in mitochondria, but most of the cycle’s

metabolites are present in both mitochondria and cytosol. Therefore it is difficult to establish

the rate-determining steps.- However, three of the eight steps have significantly negative physiological free energy

changes. The enzymes involved in those steps are likely to function far from equilibriumunder physiological conditions.

Standard ( ∆∆∆∆G°’ ) and physiol ogical ( ∆∆∆∆G) f ree energy changes Reaction Enzyme ∆G°’ (kJ/mol) ∆G (kJ/mol)

1 Citrate synthase -32.2 Negative2 Aconitase +13.3 ~03 Isocitrate dehydrogenase -20.9 Negative4

α-Ketoglutaratedehydrogenase-33.5 Negative

5 Succinyl-CoA synthetase -2.9 ~06 Succinate dehydrogenase 0.0 ~07 Fumarase -3.8 ~08 Malate dehydrogenase +29.7 ~0

- Unlike enzymes in glycolysis and glycogen metabolism, the citric acid cycle is largelyregulated by1. substrate availability (rate of diffusion of substrate into mitochondria)2. product inhibition. (NADH, ATP, citrate)

3. competitive feedback inhibition by intermediates further along the cycle.


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Products and NADH are involved in feedback i nhi bition .- ADP and ATP are allosteric regulators of isocitrate dehydrogenase. High [ADP] activates

the enzyme whereas high [ATP] inhibits the enzyme.- Ca2+ activates pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate

dehydrogenase.


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5. THE AMPHIBOLIC NATURE OF THE CITRIC ACID CYCLE - In the muscle, the citric acid cycle works mainly degradation of acetyl-CoA to produce

bioenergies (ATP).- In the liver, the citric acid cycle is amphibolic.

Note: Amphibolic = both anabolic and catabolic processes.

Anabolism: %

Catabolism: %

I ntermediates of citr ic acid cycle are also vari ous precursors

Amino acidsSugarsFatty acids, etc.

Proteins Nucleic acidsLipids, etc.

Energy yieldingmaterials, suchas proteins

Energy poor end products, such asCO2, NH3, H2O


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- Intermediates of citric acid cycle are also precursors of:- Glucose biosynthesis.- Lipid biosynthesis including fatty acid and cholesterol.

Note: Lipid biosynthesis is taken place in cytosol, but the mitochondrial acetyl-CoA(processor) cannot be transported across the inner mitochondrial membrane. Thus, acetyl-

CoA is converted to citrate by ATP-citrate lyase since citrate can cross the membrane.Why citrate synthase is not used? --- Because no ATP is produced.

ADP + Pi + oxaloacetate + acetyl-CoA ↔ ATP + citrate + CoA

- Amino acid biosynthesisα-ketoglutarate + NAD(P)H + NH4

+ ↔ Glu + NAD(P)+ + H2Oα-ketoglutarate + Ala ↔ Glu + pyruvateOxaloacetate + Ala ↔ Asp + pyruvate

- Porphyrin biosynthesis- utilizes succinyl-CoA as a starting material.

When the citric acid cycle intermediates are transported too much as precursors, theconcentration of oxaloacetate is very low. In this case, it is necessary to replenish citric acidcycle intermediates. The main reaction is:

- Pyruvate + CO2 + ATP + H2O ↔ oxaloacetate + ADP + Pi

The citr ic acid cycle is tru ly at the center of metabolism - Reduced products: NADH and FADH2 are reoxidized to produce ATP.- The citric acid intermediates are utilized in the biosynthesis of many vital cellular constituents.

crystal.res.ku.edu taksnotes biol 638 notes chp 16

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