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2. The Citric Acid Cycle (CAC)
Pyruvate
CO2
2. The Citric Acid Cycle (CAC)
The sequence of events:
• 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
2. The Citric Acid Cycle (CAC)
List of enzymes involved:
1. Synthase
 Catalyzes a synthesis process
2. Aconitase
 A stereo-specific isomerization
3. Dehydrogenase
 Removes hydrogen as H2
4. Synthetase
 Links two molecules by using the energy of
cleavage of a pyrophosphate group
5. Fumarase
 Catalyzes reversible hydration/rehydration of
fumarate to malate
Step 1: C-C Bond Formation by Condensation
of Acetyl-CoA and Oxaloacetate
Citrate Synthase
Reaction• Condensation of acetyl-CoA and oxaloacetate
• The only reaction with C-C bond formation
• Rate-limiting step of CAC
Mechanism• Uses Acid/Base Catalysis
– Carbonyl of oxaloacetate is a good electrophile
– Methyl of acetyl-CoA is NOT a good nucleophile but
is activated by deprotonation
Highly thermodynamically favorable/irreversible
– Regulated by substrate availability and product inhibition
– Activity largely depends on [oxaloacetate]
Induced Fit in the Citrate Synthase
Citrate Synthase has two subunits that create two binding
sites for binding both oxaloacetate and acetyl-CoA. Binding
is very conformation dependent:
A. Open conformation
 Free enzyme does not have a binding site for acetyl-CoA
B. Closed conformation
 Binding of OAA enables binding for acetyl-CoA
 The conformation avoids hydrolysis of thioester in acetyl-CoA
 Protects reactive carbanion
Induced Fit in the Citrate Synthase
Mechanism of Citrate Synthase:
Acid/Base Catalysis
Mechanism of Citrate Synthase:
Acid/Base Catalysis
Mechanism of Citrate Synthase:
Hydrolysis of Thioester
Step 2: Isomerization by Dehydration/
Rehydration
Aconitase
Key points:
• 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.
Aconitase is stereospecific
Only R-isocitrate is produced by aconitase
Distinguished by three-point attachment to the active site
Aconitase is stereospecific
• Only R-isocitrate is produced by aconitase because citrate is
prochiral with respect to binding to the active site.
-Distinguished by three-point attachment to the active site
Step 3: Oxidative Decarboxylation #2
Isocitrate Dehydrogenase
Key points:
• Oxidative decarboxylation
– Lose a carbon as CO2
– Oxidation of the alcohol to a ketone
– Transfers a hydride to NAD+ generating NADH
• Cytosolic isozyme uses NADP+ as a cofactor
• Highly thermodynamically favorable/irreversible
– Regulated by product inhibition and ATP
Mechanisms of Isocitrate Dehydrogenase:
Metal Ion Catalysis (Oxidation)
0
+2
Mechanisms of Isocitrate Dehydrogenase:
Metal Ion Catalysis (Decarboxylation)
Carbon lost as CO2 did NOT come from acetyl-CoA.
Mechanisms of Isocitrate Dehydrogenase:
Rearrangement and Product Release
Step 4: Final Oxidative Decarboxylation
-Ketoglutarate Dehydrogenase
Key points:
• Last oxidative decarboxylation
– Net full oxidation of all carbons of glucose
• 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
-Ketoglutarate Dehydrogenase
• Complex similar to pyruvate dehydrogenase
– Same coenzymes, identical mechanisms
– Active sites different to accommodate different-sized substrates
Origin of C-atoms in CO2
COOH
H2C
COOH
C
COOH
HC
COOH
H2C
COOH
C
H
COOH
H2C
HO
Citrate
HO
Isocitrate
H2C
COOH
H2C
CH2
O
C
COOH
-ketoglutarate
COOH
CH2
O
C
SCoA
Succinyl-CoA
Both CO2 carbon atoms derived from oxaloacetate.
 At this point in the metabolic pathway, a
total of 6 CO2 are produced.
Step 5: Generation of GTP through Thioester
Succinyl-CoA Synthetase
Key points:
• 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
Mechanism of Succinyl-CoA Synthetase
GTP Converted to ATP
• Catalyzed by nucleoside diphosphate kinase.
Step 6:Oxidation of an Alkane to Alkene
Succinate Dehydrogenase
Key points:
• Bound to mitochondrial inner membrane
– Part of Complex II in the electron-transport chain
• Reduction of the alkane to alkene (reverse reaction)
requires FADH2
– Reduction potential of NAD is too low
• FAD is covalently bound, which is unusual
• Near equilibrium/reversible
– Product concentration kept low to pull forward
Step 7: Hydration Across a Double Bond
Fumarase
Key points:
• Stereospecific
– Addition of water is always trans and forms L-malate
– OH- adds to fumarate and 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
Stereospecificity of Fumarase
Step 8: Oxidation of Alcohol to a Ketone
Malate Dehydrogenase
Key points:
• 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
3. One Turn of the Citric Acid Cycle
3A. 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
3B. Direct and Indirect ATP Yield