Citrate Synthase - Mechanism

Mechanism

Citrate Synthase has three key amino acids in its active site which catalyze the conversion of acetyl-CoA (H₃CCO-SCoA) and oxaloacetate (COO-CH₂COCOO-) into citrate (COO-CH₂COHCOOCH₂COO-) and H-SCoA in an aldol condensation reaction. This conversion begins with the negatively charged oxygen in Asp375’s R-group deprotonating acetyl CoA’s alpha carbon. This pushes the e- to form a double-bond with the carbonyl carbon, which in turn forces the C=O up to pick up a proton for the oxygen from one of the nitrogens in the R-group of His274. This neutralizes the R-group (by forming a lone pair on the nitrogen) and completes the formation of an enol intermediate (CH₂COH-SCoA). At this point, His274’s amino lone pair formed in the last step attacks the proton that was added to the oxygen in the last step. The oxygen then reforms the carbonyl bond, which frees half of the C=C to initiate a nucleophilic attack to oxaloacetate’s carbonyl carbon (COO-CH₂COCOO-). This frees half of the carbonyl bond to deprotonate one of His320’s amino groups, which neutralizes one of the nitrogens in its R-group. This nucleophilic addition results in the formation of citroyl-CoA (COOCH₂CHCOOCH₂COHSCoA2-). At this point, a water molecule is brought in and is deprotonated by His320’s amino group and Hydrolysis is initiated. One of the oxygen’s lone pairs nucleophilically attacks the carbonyl carbon of citroyl-CoA. This forms a tetrahedral intermediate and results in the ejection of –SCoA as the carbonyl reforms. The –SCoA is protonated to form HSCoA. Finally, the hydroxyl added to the carbonyl in the previous step is deprotonated and citrate (-COOCH₂COHCOO-CH₂COO-) is formed.

This link connects to a video demonstrating citrate synthase's mechanism from Lehninger's Principles of Biochemistry page.