Oxaloacetate first binds to Citrate Synthase, which induces a conformational change that creates a binding site for the second substrate, acetyl-CoA.

Part of the catalytic efficiency of the enzyme results from electrostatic destabilization of the oxaloacetate. This is accomplished by making salt bridges with two Arginine residues(329, and 401)and H-bonding to two Histidine residues (238, and 320). The His320 must be protonated for the condensation reaction to take place, so in effect the oxaloacetate is surrounded by at least three positively charged EWGs.

Now that oxaloacetate is bound in place, Acetyl-CoA enters the active site. Acetyl-CoA is a H-bond donor to Asp375 and a H-bond acceptor of His 274. In the first step of the reaction mechanism, His274 protonates the carbonyl oxygen, while Asp375 simultaneously abstracts a methyl proton. This produces a neutral enol without ever forming a high-energy charged intermediate. This step is further catalyzed by the activation of the methyl H's by the Sulfur linkage to CoA.

With the enol formed, condensation can take place. Attack on the carbonyl of oxaloacetate by the enol, followed by protonation of the carbonyl oxygen by His320, and deprotonation of the carbonyl oxygen by His274 generates the citronyl-CoA intermediate. Hydrolysis of the sulfur bond creates the target molecule citrate, and recycles Co-A.