[7] Citrate synthase catalyzes the condensation reaction of the two-carbon acetate residue from acetyl coenzyme A and a molecule of four-carbon oxaloacetate to form the six-carbon citrate:[5] Oxaloacetate is regenerated after the completion of one round of the Krebs cycle.

Citrate synthase's 437 amino acid residues are organized into two main subunits, each consisting of 20 alpha-helices.

[8] The adjacent images display the tertiary structure of citrate synthase in its opened and closed form.

[10] 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.

The inhibition of citrate synthase by acetyl-CoA analogues has also been well documented and has been used to prove the existence of a single active site.

These experiments have revealed that this single site alternates between two forms, which participate in ligase and hydrolase activity respectively.