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# TCA Cycle: Citrate Synthase Reaction Mechanism

BIOCHM-REFPU1

The first enzyme of the TCA Cycle, citrate synthase, catalyzes the aldol condensation of oxaloacetate (OAA) and acetyl-CoA to form citrate and reduced coenzyme-A. This is a highly exergonic reaction (${ \Delta G }^{ \prime °}=-32.2 kJ/mol$) due to the controlled hydrolysis of the thioester bond in the transient intermediate citroyl-CoA.

Shown below is the reaction mechanism for citrate synthase.

Use this diagram and your understanding of general enzyme catalysis to identify which of the following statements does NOT accurately describe the reaction mechanism.

A

In Step 1, Asp375 acts as a base and abstracts a proton from the methyl group of acetyl-CoA forming a reactive enolate intermediate stabilized by His274 through proton abstraction (shown in Step 2).

B

In Step 2, the enolate (formed by deprotonation in Step 1) acts as an electrophile in attacking the carbonyl carbon of oxaloacetate and forms a new $C-C$ bond (shown in Step 3).

C

In Step 2, His274 acts as a general base and His320 acts as a general acid.

D

In Step 3, the high-energy thioester intermediate citroyl-CoA is hydrolyzed following the nucleophilic attack by water on the carbonyl carbon yielding citrate and reduced Coenzyme-A.

E

High intracellular pH (greater than about 8) would inhibit this reaction mechanism by deprotonating key His residues.

F

Exposure of the reaction intermediates to water prior to citroyl-CoA formation would likely result in inhibition of citrate formation due to premature hydrolysis of the thioester bond in acetyl-CoA.