An important group of enzymes catalyse the deprotonation of weakly acidic carbon atoms adjacent to carbonyl groups. The mechanisms of such enzymes have been puzzling because the instability of postulated reaction intermediates appears inconsistent with the observed rapid rates of reaction. Citrate synthase is an enzyme of this type, which removes a proton from acetyl-CoA in a reaction step which is believed to be rate-limiting for the production of citrate. The mechanism of this reaction has been investigated with a quantum mechanical/molecular mechanical (QM/MM) technique, which allows the energy profile for the reaction in the enzyme, including the effects of the protein environment, to be calculated. The method has been tested by comparison with experimental and theoretical studies. In conjunction with structural and biochemical data, the results shed light on the mechanism of citrate synthase, in particular on the roles of the catalytic residues and the nature of the reaction intermediate. In agreement with previous proposals, Asp-375 is found to act as the base to deprotonate acetyl-CoA. The resulting enolate is intrinsically unstable, but is stabilized by hydrogen bonds from His-274, which is neutral, and a conserved water molecule. The nucleophilic character of the enolate is found to be important for the next stage of the reaction, condensation with oxaloacetate, to proceed rapidly. The energetic contributions of individual residues along the reaction pathway have been evaluated. The effective stabilization provided by hydrogen bonds is found to be dependent on the active site environment.
