A model of the condensation step in the citrate synthase reaction

Abstract

Calculations have been performed on the nucleophilic addition of simple molecules representing a thioester enolate and enol to acetone, as models for the addition step in the Claisen-type condensation reaction of acetyl-CoA and oxaloacetate catalysed by the enzyme citrate synthase. The structures of the transition state and addition product for the enolate model have been optimized at the semi-empirical (AM1, PM3 and MNDO) and ab initio ( RHF/6–31+ G( d) and MP2/6–31+ G( d)) levels. The relative energies of the transition state and addition product given by AM1 are reasonably close to the best ab initio result. The results are compared with AM1 results for addition of the enol form of the thioester model, which has been proposed as an alternative possibility for the nucleophile in the enzyme reaction. The barrier to the addition reaction for the enol model is considerably larger, due to the expected poorer nucleophilicity of the neutral enol form. It appears that the barrier for enol addition, unless significantly lowered by the enzyme, is inconsistent with the observed rate of reaction. In agreement with studies of the enolization of acetyl-CoA in citrate synthase, the enolate of acetyl-CoA appears to be a more likely nucleophilic intermediate than the enol. This conclusion may also be relevant for other enzymic Claisen condensation reactions in that efficient reaction may require stabilization of the thioester enolate to maintain a high degree of enolate character in the reactive intermediate.