Enzymes

Enzymes

• Enzymes are usually proteins which act as biological catalysts for metabolic reactions

• Enzymes enhance the rate at which the chemical reactions of a cell take place. They do not affect the equilibrium constant.

Mechanism of Enzyme Action

• Enzymes interact with reactants at specific places in the enzyme structure termed the active site.

• The enzyme is specific for a given substrate.

• The enzyme substrate complex is then converted to product which is released.

Enzyme Substrate Complex

E + S <----->ES <----------> P

Thus in a closed system free enzyme availability limits the rate of the reaction in the presence of saturating amounts of substrate.

Enzyme Substrate Complex

Fig. 8.16 p.160

Enzyme Kinetics

• Vmax = maximum rate an enzyme reaction can occur at saturating substrate concentration.

• Km = the substrate concentration which gives 1/2 Vmax

Lowering the Activation Enzymes Work by Energy of a Reaction

• Even exergonic chemical reactions do not occur spontaneously but require enough energy input to raise the reactants to a “transition state”.

• Transition state in between the free state of reactants and free state of products.

Activation Energy

Enzymes and substrate interaction

Induced Fit Model• The induced fit model says that the

specific site for substrate is a very close fit but not exact.

• When the substrate hits the active site it binds and induces a change in the protein which positions and stresses the substrate so that a reaction is most likely to occur.

Enzymes As Catalysts

Enzymes work by:

• Concentrating them at the active site

• Poising the substrate to lower the activation energy and correctly orient them in a position most likely to allow for a reaction.

Enzymes lower energy of activation

Influence of Environment on Enzyme Activity

• Effect of Temperature. The Arrhenius Plot.

Enzyme reactions will double in rate for every 10C increase in temperature until they reach an optimum.

• Effect of pH. Most enzymes have an optimal pH between 7 and 8.

Effect of pH and temperature on enzyme activity

Enzyme Composition

Enzymes which are made only of protein

• Those whose activity is optimal with only substrate

• Those which require external cofactors to be fully active: Cofactors include such things as Mg+2 or Mn+2, NAD,FAD, Zn+1, etc

Enzyme Composition

Enzymes which contain organic or inorganic molecules covalently bound to proteins.

• Apoenzyme describes the protein part of such an enzyme

• Prosthetic group is the non protein component

• Holoenzyme is the entire complex

Enzyme Composition

Examples of Holoenzymes• Cytochromes. Heme = prostetic group.• Aconitase. Enzyme of the TCA cycle.

FeS is the prosthetic group• Alpha Ketoglutarate dehydrogenase

and pyruvate dehydrogenase. Dihydrolipoic Acid is the prosthetic group

Allosteric enzyme regulation

Examples of allosterism, aspartate carbamoyltransferase and carbamoyl phosphate synthetase

Metabolism

• Solute Transport• Catabolism-Breaking complex

molecules into simple molecules to generate biological energy

• Anabolism-Building macromolecules (lipids, protein, nucleic acids and polysaccharides) from simple molecules. Biosynthesis

Transport

• Simple Diffusion: gases, water

• Facilitated or passive transport: glycerol

• Active transport: most solutes

• Group translocation: sugars including glucose and other sugars such as fructose, mannose, maltose and sucrose.

Transport

Simple Diffusion• No protein carrier molecule and therefore

does not obey saturation kinetics• No biological energy requirment• Does not work against a concentration

gradient• Examples: water (osmosis), gases.

Facilitated Diffusion

• Requires a protein carrier thus obeys saturation kinetics

• No biological energy requirement

• Cannot move solute against a concentration gradient

• Example : glycerol transport

Transport by facilitated diffusion and passive diffusion`

Facilitated Diffusion

Active Transport

• Requires metabolic energy

• Requires protein carrier and thus obeys saturation kinetics

• Moves solute against a concentration gradient

• Example: amino acid transport systems

Specific Active Transport Systems of E. coli

Binding Protein Transport Systems• Metabolic energy source is ATP directly• Binding proteins located in periplasmic space

bind substrate• Substrate carrying binding proteins lock with

cytoplasmic membrane carriers which transport substrate using ATP as energy. Histidine transport is an example.

Gradient Driven Active Transport

• Metabolic energy used is usually proton motive force (H+ gradient) established through respiration

• No binding protein but has transmembrane carrier protein

• Systems incude Antiport, Symport and uniport. Examples, sodium export, lactose transport and nitrite export

Antiport and symport

Group TranslocationGroup Translocation

Phosphoenolpyruvate:sugar phosphotransferase system

§ Requires no net metabolic energy§ Requires protein carrier as well as

cytoplasmic proteins§ Solute is modified during transport

and therefore cannot discuss concentration gradients

Phosphoenolpyruvate:sugar phosphotransferase system

§ Requires no net metabolic energy§ Requires protein carrier as well as

cytoplasmic proteins§ Solute is modified during transport

and therefore cannot discuss concentration gradients

Phosphoenolpyruvate:Sugar Phosphotransferase System (PTS)

Phosphoenolpyruvate:Sugar Phosphotransferase System (PTS)

Microbe of the Week

Salmonella typhiGram-negative, motile, mesophilic enteric bacterium

Causative agent of typhoid fever (aka “enteric fever”

Typhoid fever: the illness

7-28 days (avg. 14 days)Fever, malaise, anorexia, spots on trunkDiarrhea or constipationDelirium75% hospitalizedFatality rate = 0.4%Recovery: 1-8 weeks

Sources

Humans are sole reservoir (does not infect animals)Carriers may harbor the organism in their gall bladderContaminated food – by handlers (milk, sandwiches, meat, cake!)

or …Contaminated water – e.g. shellfish in polluted watersOrganism survives in shellfish up to 4 days, sea water up to 9 days, for weeks in sewageTransmission: mainly from water contaminated with human waste or human carriers

Typhoid MaryTyphoid MarySociological implications of infectious disease

Typhoid Mary's real name was Mary Mallon. Irish immigrant who made her living as a cookMallon was the first person found to be a "healthy carrier" of typhoid fever in the United States.

She herself was not sick – but over 30% of the bacteria in her feces were S. typhi

Mallon is attributed with infecting 47 people with typhoid fever, three of whom died.Interred on a N. Brother Island, NY for 26 years

1907-1910 1915- till her death in 1938

Sociological Implications of Infectious DiseaseTyphoid Mary

Mary Mallon (wearing glasses) photographed with bacteriologist Emma Sherman on North Brother Island in 1931 or 1932, over 15 years after she had been quarantined there permanently.

ENERGY GENERATION

ENERGY GENERATION

The Two General Mechanisms for Making Energy

• Substrate Phosphorylation: ATP is made directly through a specific enzymatic exergonic reaction. Examples: 3 phosphoglycerate kinase and pyruvate kinase.

• Respiratory driven proton translocation coupled with ATP synthesis otherwise known as the Chemiosmotic Mechanism

Substrate Phosphorylation

General Concept

A + B + ADP + Pi<--------->C + D + ATP

Usually specific reactions of glycolysis are given as examples of these kinds of

reactions

Chemiosmotic Theory

Couples respiration with a proton gradient that can be used to drive ATP synthesis

through the ATPase enzyme

Fig.9.11 p.172

Fig. 9.1 p.165

Fig.9.3 p.166

Fig.

ATP requiring and ATP yielding reactions in glycolysis

ATP requiring and ATP yielding reactions in glycolysis

Requiring ATP1. Glucose + ATP------->glucose-6P + ADP

(6C) Hexokinase (6C)2. Fructose-6P + ATP----->Fructose-1,6 bisphosphate +ADP

(6 carbons) phosphofructokinase (6carbons)Yielding ATP

1. 1,3 bisphosphoglycerate+ADP----->1phosphoglycerate + ATP

(3Carbons) phosphoglycerate kinase (3Carbons)

z Phosphoenolpyruvate + ADP-----------> Pyruvate + ATP (3Carbons) pyruvate kinase (3Carbons)

1. Net Gain from substrate phosphorylation per glucose=2ATP

Requiring ATP1. Glucose + ATP------->glucose-6P + ADP

(6C) Hexokinase (6C)2. Fructose-6P + ATP----->Fructose-1,6 bisphosphate +ADP

(6 carbons) phosphofructokinase (6carbons)Yielding ATP

1. 1,3 bisphosphoglycerate+ADP----->1phosphoglycerate + ATP

(3Carbons) phosphoglycerate kinase (3Carbons)

z Phosphoenolpyruvate + ADP-----------> Pyruvate + ATP (3Carbons) pyruvate kinase (3Carbons)

1. Net Gain from substrate phosphorylation per glucose=2ATP

Specific Glycolytic Reaction forming Reduced NAD

Glyceraldehyde 3 phosphate + NAD + Pi<------->1,3 bisphosphate glycerateglyceraldehyde 3 phosphate dehydrogenase

Generation of Electron Donors for RespirationGeneration of Electron Donors for Respiration

l The two primary sources of electrons The two primary sources of electrons for the respiratory chain come from for the respiratory chain come from NADHNADH++ or FADH or FADH++

l These reducing equivalents come These reducing equivalents come primarily from the final degradation of primarily from the final degradation of glucose via the TCA cycleglucose via the TCA cycle

l The two primary sources of electrons The two primary sources of electrons for the respiratory chain come from for the respiratory chain come from NADHNADH++ or FADH or FADH++

l These reducing equivalents come These reducing equivalents come primarily from the final degradation of primarily from the final degradation of glucose via the TCA cycleglucose via the TCA cycle

Pyruvate Dehydrogenase RXPyruvate Dehydrogenase RX

Pyruvate + CoA + NAD ---------> AcetylCoA +NADH2 +CO2

*First decarboxylation after glycolysis before TCA*Generates NADH *Generates the AcetylCoA which then enters TCA

Fig.9.7 p.170

Specific Reactions of TCA forming Reduced NAD and FAD

• Isocitrate + NAD <--isocitrate dehydrogenase-> alpha ketoglutarate + NADH2 + CO2

• Alpha ketoglutarate + NAD<---------------->succinate CoA + NADH2 + CO2

alpha ketoglutarate dehydrogenase

• Succinate + FAD<------------------------------>fumarate + FADH2

succinate dehydrogenase

• Malate + NAD<-------------------------------------> oxaloacetate + NADH2

malate dehydrogenase

RespirationRespiration

s The respiratory chain is composed of electron carriers ordered by redox potential from more negative to more positive

s The initial source of electrons is NADH+ or FADH+

s Initially two electrons and two protons enter the respiratory chain and are passed according to redox potential with the ultimate reduction of oxygen to water.

s The respiratory chain is composed of electron carriers ordered by redox potential from more negative to more positive

s The initial source of electrons is NADH+ or FADH+

s Initially two electrons and two protons enter the respiratory chain and are passed according to redox potential with the ultimate reduction of oxygen to water.

Components of Respiratory Chain

Components of Respiratory Chain

NADH dehydrogenaseSuccinate dehydrogenase

(flavoprotein)Quinones (organic molecule)Cytochromes a, b, c and

cytochrome oxidase (a, d, o)

NADH dehydrogenaseSuccinate dehydrogenase

(flavoprotein)Quinones (organic molecule)Cytochromes a, b, c and

cytochrome oxidase (a, d, o)

Fig. 9.9 p.171

Fig. 9.10 p. 172

Inhibitors of Respiration

• Inhibitors of respiration specifically block the transfer of electrons from one specific respiratory carrier to the other.

• These inhibitors do not affect the permeability of the membrane and a PMF can still be maintained through the hydrolysis of ATP

Examples of Inhibitors of Respiration

• Antimycin blocks electron transport between cytochrome b and cytochrome c

• Cyanide and Azide prevent the transfer of electrons to oxygen e.g. they inhibit cytochrome oxidase.

Fig. 9.11 p. 172

Uncouplers of Oxidative Phosphorylation

• Uncouplers basically make membranes permeable to ions such as H+ thus the PMF collapses and can’t support ATP synthesis.

• Most uncouplers cause the cell to increase oxygen respiration rate

• Examples of uncouplers are dinitrophenol (DNP) , nigericin and valinomycin.

Fig. 9.12 p.173

Respiration/ATP stoichiometry

• Per molecule of phosphate and 1/2 O2

microoganisms generate a certain amount of ATP. This is called a P/O ratio and is usually 3/1 in mitochondria

• The ratio is thought to be lower in E. coli

Summary

• NADH2<------->NAD yields 3 ATP in mitochondria

• FADH2<----------->FAD yields 2 ATP in mitochondria

• From glucose to CO2, 10 NADH2 and 2 FADH2 are formed. Therefore 34ATP formed from O/P and 4 from SP.

Table 9.2

Generation of Energy Under Anaerobic Conditions

• Fermentation: Energy is generated strictly by substrate level phosphorylation (SLP). NADH2 is recycled to NAD by specific fermentation reactions.

• Anaerobic Respiration: Energy is generated in the same way as aerobic respiration but with lower energy yields. NADH2 is recycled to NAD through an anaerobic respiratory chain.

Specific Reactions Forming Substrate Level ATP

Glycolysis

• 1,3 bisphosphate glycerate +ADP<---------->3 phosphoglycerate +ATP

3 phosphoglycerate kinase

• Phosphoenolpyruvate + ADP<------------------> pyruvate + ATPpyruvate kinase

Specific Glycolytic Reaction forming Reduced NAD

• Glyceraldehyde 3 phosphate + NAD + Pi<------->1,3 bisphosphate glycerate

glyceraldehyde 3 phosphate dehydrogenase

Fermentation is an Anaerobic Process

• Necessary to limit reactions that generate NADH2 and to develop specific reactions to regenerated NAD so glycolysis can continue.

• ATP formed strictly from SLP.

Fermentation Strategy

• Fermentation reactions are aimed at converting the NADH2 to NAD

• Intermediates of the TCA cycle are necessary for biosynthesis. A split TCA cycle so that there is a limitation of NADH2made.

• Formation of Acetyl CoA is by Pyruvate formate lyase so that NADH2 is not formed

Fermentation Reactions

• Microorganisms have developed diverse reactions to regenerate NAD for glycolysis

• Strategy is to use organic compounds as electron and proton acceptors for the oxidation of NADH2

Examples of Fermentation Reactions

• Pyruvate + NADH2 <-------->Lactate + NAD

lactate dehydrogenase

• Pyruvate + <--------> acetaldehyde + CO2

acetaldehyde + NADH2<------->ethanol + NAD

alcohol dehydrogenase

TCA cycle During Fermentation

• The TCA cycle has a both catabolic and anabolic functions.

• During biosynthesis intermediates of the TCA cycle are drawn off to provide carbon skeletons for important biosynthetic pathways

• The TCA cycle is modified during fermentation to limit production of NADH2

TCA Cycle Is Modified During Fermentation

• Split TCA cycle limits amount of NADH2

formed• The enzyme alpha ketoglutarate

dehydrogenase is not made under fermentation conditions

• The last half of the cycle runs backwards

• Thus all intermediates are made but NADH2 production is limited

Split TCA

• Pyruvate + CO2 <-----> Oxaloacetate

• 1/2 of the Oxaloacetategoes to form citrate and 1/2 to the backward reactions of TCA

Fermentation End Products of Commercial Value

• Alcohol (wine, beer, distilled beverages)

• Amino acids

• Acetone

• Butanol

• Citrate

• 2,3 butanediol for butter flavor

Anaerobic Respiration

• Some but not all bacteria can also grow anaerobically using compounds other than oxgen as terminal electron acceptors for respiration.

• Examples of anaerobic terminal electron acceptors are NO3

-, and SO4-.

• NO3- respiration is by far the most

common

Anaerobic Respiration

• Catabolic pathways are similar to aerobic pathways

• NADH2 and FADH2 are formed in the same way as aerobically (glycolysis and TCA)

• NADH2 and FADH2 are oxidized by the appropriate specialized anaerobic respiratory chain located in the membrane

Energy Generation during Anaerobic Respiration

• Energy is generated by the same mechanisms as during aerobic respiration.

• Because the redox potential of the terminal electron acceptors are not as positive as the oxygen/water couple the PMF generated is not as strong and thus per 2 electrons passed down the chain less ATP can be made through the chemiosmotic mechanism.

Photosynthesis

Two sets of reactions

• Light reactions: light energy is trapped and converted to chemical energy

• Dark reactions: the energy generated during the light reaction is used to fix CO2 to sugar.

Bacterial Photosynthesis

• Blue green algae perform eucaryotic like photosynthesis which is oxygenic

• All other eubacterial photosynthesis is anoxygenic i.e. does not generate oxygen.

Oxygenic Photosynthesisblue green algae

• Occurs in specialized organelles called chloroplasts which contain the chlorophylls and other pigments that absorb light energy

• Light reactions occur in the thylakoid membrane (inner membrane) of the chloroplast

• Dark reactions are in the fluid surrounding the thylakoid membranes called stroma

Plant photosynthesis

Carbon Dioxide Fixation

• Requires lots of reducing power (NADH and NADPH)

• Occurs via the Calvin Cycle in both bacterial and eucaryotic photosynthesis

4 families of anaerobic photosynthetic bacteria

• Purple non sulfur bacteria• Purple sulfur bacteria• Green sulfur bacteria• Green gliding bacteriaThese organisms do anoxygenic

photosynthesis and use something other than water as a source of electrons for reducing equivalents

Reduction potential in anaerobic photsynthetic bacteria

1. Reverse Electron Flow

2. Direct shuttle via specific enzymes from inorganic and Organic molecules such as H2S or H2

Bacterial anaerobic photosynthesis

• Cyclic photosynthesis

• Reducing power usually from reverse electron flow or directly from inorganic or organic compounds

• CO2 fixation via Calvin Cycle

Chemolithotrophic Metabolism

• Energy source is an inorganic compound

• Carbon source is CO2

• Reducing power for biosynthesis comes from organic or inorganic compounds such as succinate, malate or sulfur.

Examples of Chemolithotrophic Activity

Nitrification

• Ammonia------------------->NitriteNitrosomonas

• Nitrite------------------------>NitrateNitrobacter

Chemolithotrophic autotrophNitrobacter

BIOSYNTHESIS

Building Blocks for Macromolecules

• Amino acids (AA)--------------->proteins

• Glycerol + fatty acids-------------->lipids

Glycerol + fatty acids +AA + Pi--->phospholipids

• Monosaccharides--------->polysaccharides

• Pyrimidines +purines +nitrogen +riboseP---->nucleic acids

Metabolic Pathways Providing Intermediates of Metabolism for The Synthesis for Macromolecular

Building Blocks

• Embden Myerhoff Parnas (Glycolysis)

• Tricarboxylic Acid Cycle (Krebs)

• Pyruvate Dehydrogenase reaction

Anaplerotic Reactions

Fill-in intermediates of TCA that are drawn-off for biosynthesis so cycle can

continue

• PEP + CO2-------> Oxaloacetate

• Pyruvate + CO2------->Oxaloacetate

• Glyoxylate by-pass

Sources of Nitrogen, and Sulfur for Amino Acids and Nucleotides

• Organic nitrogen and sulfur containing compounds. Amino Acids (20), pyrimidine and purine bases (ATUGC) from the breakdown of protein and nucleic acids.

• Assimilation of inorganic nitrogen and sulfur compounds (N2, NH4

+, NO3-, SO4

-2)

Peptidoglycan Synthesis

• NAG and NAM-peptide are made in cytoplasm. The peptide is synthesized via a non ribosomal enzyme system.

• The above are then transported across the membrane by proteins called bactoprenols

Antibiotics Interfering with Peptidoglycan Synthesis

• Penicillin and vancomycin inhibit transpeptidation

• Cycloserine inhibits the cytoplasmic synthesis of the pentapeptide unit

• Bacitracin interfers with the transport of NAM-peptide and NAG through the membrane by bactoprenol.