2. The Citric Acid Cycle (CAC) Pyruvate CO 2

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 FADH 2 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 H 2 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 H 2 O 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 H 2 O 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 CO 2 –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) +2 0

Mechanisms of Isocitrate Dehydrogenase: Metal Ion Catalysis (Decarboxylation) Carbon lost as CO 2 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 CO 2 Both CO 2 carbon atoms derived from oxaloacetate.  At this point in the metabolic pathway, a total of 6 CO 2 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 FADH 2 –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 Net oxidation of two carbons to CO 2 –Equivalent to two carbons of acetyl-CoA –but NOT the exact same carbons Energy captured by electron transfer to NADH and FADH 2 Generates 1 GTP, which can be converted to ATP Acetyl-CoA + 3NAD + + FAD + GDP + P i + 2 H 2 O  2CO 2 + 3NADH + FADH 2 + GTP + CoA + 3H +

3B. Direct and Indirect ATP Yield