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.
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