chapter 16 Flashcards
Fates of Pyruvate
fermentation to ethanol in yeast
-pyruvate->acetyl-coA->citric acid cycle, under aerobic conditions
-fermentation to lactate in vigorously contracting muscle, in erythrocytes, in some other cells, and in some microorganisms
Respiration & Cellular Respiration
-Process in which cells consume O2 and produce CO2
-Provides more energy (ATP) from glucose than glycolysis
-Also captures energy stored in lipids and amino acids
-Evolutionary origin: developed about 2.5 billion years ago (later than glycolysis)
-Used by animals, plants, and many microorganisms
Cellular Respiration
Stage 1: Acetyl-CoA Production
Oxidation of fatty acids, glucose and some amino acids yields acetyl-CoA
Stage 2: Acetyl-CoA Oxidation
Acetyl groups are fed into the TCA cycle and oxidized CO2- the energy released is conserved in electron carriers NADH, FADH2 and GTP (mitochondrial matrix, except succinate dehydrogenase located in the inner membrane)
Stage 3: Oxidative Phosphorylation
NADH and FADH2 oxidized, H+ and e- transferred to O2, generates a lot of ATP (inner membrane)
Respiration: Stage 1
Acetyl-CoA Production
Converts pyruvate to acetyl-CoA, generates some ATP, NADH and FADH2 by using the enzyme pyruvate dehydrogenase complex
Conversion of Pyruvate to Acetyl-CoA
Net Reaction:
-Oxidative decarboxylation of pyruvate
-First carbons of glucose to be fully oxidized to CO2
Catalyzed by the pyruvate dehydrogenase complex
-Requires 5 coenzymes
-TPP, lipoyllysine, and FAD are prosthetic groups
-NAD+ and CoA-SH are co-substrates
Structure of Coenzyme A
Coenzymes are not a permanent part of the enzymes’ structure.
–They associate, fulfill a function, and dissociate
CoA-SH to emphasize the active SH group contains pantothenate. The SH group forms a thioester with the acyl group. Thioesters have a high acyl group transfer potential and can transfer the acyl group to a variety of acceptor molecules
The function of CoA is to accept and carry acetyl groups
Structure of Lipoyllysine
Prosthetic groups are strongly bound to the protein
–The lipoic acid is covalently linked to the enzyme via a lysine residue
Lipoyllysyl moiety of dihydrolipoyl transacetylase acts as a carrier of hydrogen and an acetyl group
Pyruvate Dehydrogenase Complex
(PDC)
Advantages of multienzyme complexes:
PDC is a large (up to10 MDa) multienzyme complex
-pyruvate dehydrogenase (E1)
-dihydrolipoyl transacetylase (E2)
-dihydrolipoyl dehydrogenase (E3)
Advantages of multienzyme complexes:
-short distance between catalytic sites allows -channeling of substrates from one catalytic site to another
channeling minimizes side reactions
- regulation of activity of one subunit affects the entire complex
Overall Reaction of PDC
An example of substrate channelling- intermediates do not leave the complex; [S] of E2 high; the acetyl group is not lost to other reactions.
E1- TPP
E2-Lipolysine, CoA-SH
E3-FAD, NAD
Sequence of Events in Oxidative Decarboxylation of Pyruvate
Enzyme 1
Step 1: Decarboxylation of pyruvate to an aldehyde
Step 2: Oxidation of aldehyde to a carboxylic acid
-Electrons reduce lipoamide and form a thioester
Enzyme 2
Step 3: Formation of acetyl-CoA (product 1)
Enzyme 3
Step 4: Reoxidation of the lipoamide cofactor
Step 5: Regeneration of the oxidized FAD cofactor
Forming NADH (product 2)
Respiration: Stage 2
Acetyl-CoA oxidation (TCA Cycle)
Generates more NADH, FADH2, and one GTP
Sequence of Events in the Citric Acid Cycle
Step 1: C-C bond formation to make citrate
Step 2: Isomerization via dehydration/rehydration
Steps 3–4: Oxidative decarboxylations to give 2 NADH
Step 5: Substrate-level phosphorylation to give GTP
Step 6: Dehydrogenation to give reduced FADH2
Step 7: Hydration
Step 8: Dehydrogenation to give NADH
Step 1
Formation of Citrate
C-C Bond Formation by Condensation of Acetyl-CoA and Oxaloacetate
-32.2kj/mol
-citrate synthase
Citrate Synthase
-Condensation of acetyl-CoA and oxaloacetate
-The only reaction with C-C bond formation
Uses Acid/Base Catalysis
—Carbonyl of oxaloacetate is a good electrophile
—Methyl of acetyl-CoA is not a good nucleophile…
—…unless activated by deprotonation
-Rate-limiting step of CAC
-Activity largely depends on [oxaloacetate]
-Highly thermodynamically favorable/irreversible
–Regulated by substrate availability and product inhibition
Induced Fit in the Citrate Synthase
Conformational change occurs upon binding oxaloacetate
Avoids unnecessary hydrolysis of thioester in acetyl-CoA
Open conformation:
Free enzyme does not have a binding site for acetyl-CoA
Closed conformation:
Binding of OAA creates binding for acetyl-CoA
Reactive carbanion is protected
Closed form with bound oxaloacetate and a stable analog of acetyl-CoA (carboxymethyl-CoA)
Step 2
Formation of Isocitrate via cis-Aconitate
Isomerization by Dehydration/Rehydration
=13.3kJ/mol
aconitase
Aconitase
-Elimination of H2O from citrate gives a cis C=C bond
–Lyase
-Citrate, a tertiary alcohol, is a poor substrate for oxidation
-Isocitrate, a secondary alcohol, is a good substrate for oxidation
-Addition of H2O to cis-aconitate is stereospecific
-Thermodynamically unfavorable/reversible
–Product concentration kept low to pull forward
Iron-Sulfur Center in Aconitase
Water removal from citrate and subsequent addition to cis-aconitate are catalyzed by the iron-sulfur center: sensitive to oxidative stress.
Citrate: A Symmetrical Molecule That Reacts
Asymmetrically
The two carbons brought in by acetyl-CoA are not the ones lost as carbon dioxide
Aconitase is
stereospecific
Only R-isocitrate is produced by aconitase
Distinguished by three-point attachment to the active site
Step 3
Oxidation of Isocitrate to α-Ketoglutarate and CO2
-Oxidative decarboxylation 2#
-Oxidative decarboxylation of isocitrate to make α-ketoglutarate
-isocitrate dehydrogenase
Isocitrate Dehydrogenase
Oxidative decarboxylation
–Lose a carbon as CO2
–Generate NADH
Oxidation of the alcohol to a ketone
–Transfers a hydride to NAD
Cytosolic isozyme uses NADP+ as a cofactor
Highly thermodynamically favorable/irreversible
–Regulated by product inhibition and ATP
Mechanisms of Isocitrate Dehydrogenase:
Metal Ion Catalysis (Decarboxylation)
Carbon lost as CO2 did
Carbon lost as CO2 did NOT come from acetyl-CoA.
step 4
Oxidation of α-Ketoglutarate to Succinyl-CoA, CO2
-Final oxidative decarboxylation
-Energy of oxidation is conserved in formation of a thioester bond with succinyl-CoA
-33.5kJ/mol
-alpha ketoglutarate dehydrogenase complex
α-Ketoglutarate Dehydrogenase
-Carbons not directly from glucose because carbons lost came from
-Succinyl-CoA is another higher-energy
Last oxidative decarboxylation
-Net full oxidation of all carbons of glucose
—After two turns of the cycle
—Carbons not directly from glucose because carbons lost came from oxaloacetate
-Succinyl-CoA is another higher-energy thioester bond
-Highly thermodynamically favorable/irreversible
—Regulated by product inhibition
Origin of C-atoms in CO2
Both CO2 carbon atoms derived from oxaloacetate
α-Ketoglutarate Dehydrogenase
Complex similar to pyruvate dehydrogenase
–Same coenzymes, identical mechanisms
–Active sites different to accommodate different-sized substrates
Step 5
-2.9kJ/mol
Generation of GTP through Thioester:
Thioester bond of succinyl-CoA has a ΔG’º of -36kJ/mol and is used to drive the synthesis of a phosphoanyhdride bond in ATP or GTP with a net ΔG’º of -2.9 kJ/mol
-succinyl-CoA synthetase
Succinyl-CoA Synthetase
-Substrate level phosphorylation
-Energy of thioester allows for incorporation of inorganic phosphate
-Goes through a phospho-enzyme intermediate
-Produces GTP, which can be converted to ATP
-Slightly thermodynamically favorable/reversible
–Product concentration kept low to pull forward
Step 6
Oxidation of succinate to Fumarate
Oxidation of alkane to alkene
0kJ/mol
-succinate dehydrogenase
Succinate Dehydrogenase
Bound to
Oxidation of the alkane to alkene requires
Bound to mitochondrial inner membrane
-Part of Complex II in the electron-transport chain
Oxidation of the alkane to alkene requires FAD
-Reduction potential of NAD is too low
FAD is covalently bound, unusual
Near equilibrium/reversible
-Product concentration kept low to pull forward
Step 7
Hydration of Fumarate to Malate
-3.8kJ/mol
Hydration Across a Double Bond
Fumarase
Stereospecific
-Addition of water is always trans and forms L-malate
-OH- adds to fumarate… then H+ adds to the carbanion
-Cannot distinguish between inner carbons, so either can gain –OH
Slightly thermodynamically favorable/reversible
-Product concentration kept low to pull reaction forward
Step 8
Oxidation of Malate to Oxaloacetate
Oxidation of Alcohol to a Ketone
=29.7kJ/mol
Malate Dehydrogenase
Regenerates oxaloacetate for
Oxaloacetate concentration kept
Final step of the cycle
Regenerates oxaloacetate for citrate synthase
Highly thermodynamically UNfavorable/reversible
-Oxaloacetate concentration kept VERY low by citrate synthase
—Pulls the reaction forward
One Turn of the Citric Acid Cycle
Complete oxidation of glucose is about 2840 kJ/mol. Overall energy from cellular respiration is about 32 molecules of ATP at about 30.5kJ/mol and an overall conservation of about 34% of the maximum yield.
Net Result of the Citric Acid Cycle
Acetyl-CoA + 3NAD+ + FAD + GDP + Pi + 2 H2O
2CO2 + 3NADH + FADH2 + GTP + CoA + 3H+
Net oxidation of two carbons to CO2
-Equivalent to two carbons of acetyl-CoA
-but NOT the exact same carbons
-Energy captured by electron transfer to NADH and FADH2
-Generates 1 GTP, which can be converted to ATP
-Completion of cycle
Respiration: Stage 3
Oxidative Phosphorylation
-Generates a lot of ATP
CAC intermediates are amphibolic
The cycle provides energy and also provides intermediates that are used in biosynthesis
Anaplerotic reactions (red )
Anaplerotic Reactions
-Intermediates in the citric acid cycle can be used in biosynthetic pathways (removed from cycle)
-Must replenish the intermediates in order for the cycle and central metabolic pathway to continue
-4-carbon intermediates are formed by carboxylation of 3-carbon precursors
Regulation of the Citric Acid Cycle
Two levels of regulation
Outside the TCA Cycle
-Pyruvate dehydrogenase complex
Inside the TCA Cycle
-Citrate synthase
Isocitrate dehydrogenase
α-ketoglutarate dehydrogenase
Regulation of the Citric Acid Cycle
Regulated at highly thermodynamically
Regulated at highly thermodynamically favorable and irreversible steps
-PDH, citrate synthase, IDH, and KDH
General regulatory mechanism
-Activated by substrate availability
-Inhibited by product accumulation
-Overall products of the pathway are NADH and ATP
—Affect all regulated enzymes in the cycle
—Inhibitors: NADH and ATP
—Activators: NAD+ and AMP
Regulation of Pyruvate Dehydrogenase
Pyruvate dehydrogenase complex is regulated by two different mechanisms
–Allosteric regulation
–Regulation by covalent modification
Mainly by reversible phosphorylation of E1
–Phosphorylation: inactive
–Dephosphorylation: active
PDH kinase and PDH phosphorylase are part of mammalian PDH complex
-Kinase is activated by ATP
—High ATP phosphorylated PDH less acetyl-CoA
—Low ATP kinase is less active and phosphorylase removes phosphate from PDH more acetyl-CoA
Additional Regulatory Mechanisms
Citrate synthase is also inhibited by
Regulation of isocitrate dehydrogenase controls
-Inhibition of IDH leads to
-Accumulated citrate leaves
Citrate synthase is also inhibited by succinyl-CoA
-α-ketoglutarate is an important branch point for amino acid metabolism
-Succinyl-CoA communicates flow at this branch point to the start of the cycle
Regulation of isocitrate dehydrogenase controls citrate levels
-Aconitase is reversible
-Inhibition of IDH leads to accumulation of isocitrate and reverses acconitase
-Accumulated citrate leaves mitochondria and inhibits phosphofructokinase in glycolysis
Glyoxylate Cycle
-A variation of TCA cycle, an anabolic pathway, used for the net synthesis of glucose from lipids
-occurring in plants, certain invertebrates and some microorganisms (E coli, yeast)
-Not found in vertebrates that lack isocitrate lyase and malate synthase
-Isozymes are found where there are common enzymes between the citric acid cycle and glyoxylate cycle
Glyoxylate Cycle Operates in
Glyoxysomes in Plants
Electron micrograph of a germinating cucumber seed, showing a glyoxysome, mitochondria, and surrounding lipid bodies.
Compartmentation of Glyoxylate Cycle
-Overall reaction of glyoxylate cycle:
2Acetyl-CoA + NAD+ + 2H2O → succinate + 2CoA + NADH + H+
-Each turn of the cycle consumes two molecules of acetyl-CoA and produces one molecule of succinate
-Succinate can be used for biosynthesis
Plants can synthesize glucose from acetate but mammals can’t!
Coordinated Regulation of Glyoxylate and TCA Cycles
Control point is isocitrate dehydrogenase. Phosphorylation of the enzyme which inactivates it so that glyoxylate cycle is engaged
