MODELING ENZYME REACTION INTERMEDIATES AND TRANSITION-STATES - CITRATE SYNTHASE

To investigate the roles of catalytic residues and the nature of the n ucleophilic intermediate in citrate synthase, calculations have been c arried out on models of the first stage of the reaction with ab initio (MP2/6-31+G(d)//6-31+G(d)) and semiempirical (AM1 and PM3) methods. T he first stage of the reaction involves deprotonation of acetyl-CoA, w ith Asp-375 identified as the likely base. The resulting intermediate is believed to be stabilized by a hydrogen bond from a neutral histidi ne side chain (His-274), which has been suggested to be a ''short, str ong'' or ''low-barrier'' hydrogen bond. Such bonds have been suggested to have exceptionally high energies and to stabilize many enzyme reac tion intermediates. Transition state and stable hydrogen bonded comple x geometries have been fully optimized for models of proton transfer b etween Asp-375 and acetyl-CoA and between the enolate of acetyl-CoA an d His-274. The results support the proposal that Asp-375 is the base i n the reaction and show that stabilization of the thioester enolate at the active site is required if it is to exist as a reaction intermedi ate. The effective basicities of methylimidazolate (representing His-2 74) and the thioester enolate in the hydrogen bonded complex are calcu lated to be clearly different. The enolate-methylimidazole complex (wi th the proton localized on methylimidazole) is found to be significant ly lower in energy than the enol complex (in which the hydrogen bonded proton is transferred from methylimidazole), which is not stable. Unl ess the pK(a) for deprotonation of neutral His-274 is lowered by condi tions in the active site, it appears that the hydrogen bond with the e nolate will not be of the low-barrier type. The highly favorable energ y of the interaction between the thioester enolate and methylimidazole , as well as calculations on a larger model including all three compon ents, indicate that a normal hydrogen bond with His-274 can make an im portant contribution to stabilization of the enolate intermediate.