Citrate synthase has three key amino acids in its active site (known as the catalytic triad) which catalyze the conversion of acetyl-CoA [H₃CC(=O)−SCoA] and oxaloacetate [⁻O₂CCH₂C(=O)CO₂⁻] into citrate [⁻O₂CCH₂C(OH)(CO₂⁻)CH₂CO₂⁻] and H−SCoA in an aldol condensation reaction.
 This conversion begins with the negatively charged carboxylate side chain oxygen atom of Asp-375 deprotonating acetyl CoA’s alpha carbon atom to form an enolate anion which in turn is neutralized by protonation by His-274 to form an enol intermediate [H₂C=C(OH)−SCoA].
At this point, the epsilon nitrogen lone pair of electrons on His-274 formed in the last step abstracts the hydroxyl enol proton to reform an enolate anion that initiates a nucleophilic attack on the oxaloacetate’s carbonyl carbon [⁻O₂CCH₂C(=O)CO₂⁻] which in turn deprotonate the epsilon nitrogen atom of His-320.
 This nucleophilic addition results in the formation of citroyl−CoA [⁻O₂CCH₂CH(CO₂⁻)CH₂C(=O)−SCoA].
 At this point, a water molecule is deprotonated by the epsilon nitrogen atom of His-320 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 [⁻O₂CCH₂C(OH)(CO₂⁻)CH₂CO₂⁻] is formed.
Citrate synthase has three key amino acids in its active site (known as the catalytic triad) which catalyze the conversion of acetyl-CoA [H₃CC(=O)−SCoA] and oxaloacetate [⁻O₂CCH₂C(=O)CO₂⁻] into citrate [⁻O₂CCH₂C(OH)(CO₂⁻)CH₂CO₂⁻] and H−SCoA in an aldol condensation reaction.  This conversion begins with the negatively charged carboxylate side chain oxygen atom of Asp-375 deprotonating acetyl CoA’s alpha carbon atom to form an enolate anion which in turn is neutralized by protonation by His-274 to form an enol intermediate [H₂C=C(OH)−SCoA]. At this point, the epsilon nitrogen lone pair of electrons on His-274 formed in the last step abstracts the hydroxyl enol proton to reform an enolate anion that initiates a nucleophilic attack on the oxaloacetate’s carbonyl carbon [⁻O₂CCH₂C(=O)CO₂⁻] which in turn deprotonate the epsilon nitrogen atom of His-320.  This nucleophilic addition results in the formation of citroyl−CoA [⁻O₂CCH₂CH(CO₂⁻)CH₂C(=O)−SCoA].  At this point, a water molecule is deprotonated by the epsilon nitrogen atom of His-320 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 [⁻O₂CCH₂C(OH)(CO₂⁻)CH₂CO₂⁻] is formed.
Citrate synthase