The reaction of the enzyme citrate synthase was studied using the ONIOM method, in which different layers of the system are evaluated using different levels of quantum mechanical theory. A 10 Angstrom radius model of the citrate synthase active site and surrounding protein environment was studied using MOZYME as the low level to describe the larger portion of the system. MOZYME is a semiempirical method in which the computational time scales linearly with the size of the system, enabling quantum mechanical calculations on fairly large molecular systems. Higher level ab initio methods were used to evaluate the reactive portion of the substrate and active site functionality directly involved in the reaction. This novel ONIOM-MOZYME method produced results that agree with previous studies that predict an enolate intermediate in the citrate synthase catalyzed deprotonation of acetyl-coenzyme A (acetyl-CoA). The calculated activation energy for enolate formation (14.8 kcal/mol) is very close to the experimentally determined free energy of activation. Calculated energies for an enol intermediate or a half-protonated enolic intermediate stabilized by a low-barrier hydrogen bond are 10-13 kcal/mol higher. The ONIOM-MOZYME method was also used to study the citrate synthase catalyzed deprotonation of fluoroacetyl-CoA. The results rationalize the stereoselectivity observed in the deprotonation of fluoroacetyl-CoA in the context of an enolate intermediate. This provides a reconciliation of the stereoselectivity in deprotonation of fluoroacetyl-CoA, which was previously attributed to an enol intermediate, with other studies that support an enolate intermediate for the reaction of citrate synthase. The ONIOM-MOZYME method is demonstrated to be a powerful method for the prediction of mechanism and activation energy and for analysis of structural changes in the active site during catalysis.