ACETYL-COA ENOLIZATION IN CITRATE SYNTHASE - A QUANTUM-MECHANICAL MOLECULAR MECHANICAL (QM MM) STUDY/

Citrate synthase forms citrate by deprotonation of acetyl-CoA followed by nucleophilic attack of this substrate on oxaloacetate, and subsequ ent hydrolysis. The rapid reaction rate is puzzling because of the ins tability of the postulated nucleophilic intermediate, the enolate of a cetyl-CoA. As alternatives, the enol of acetyl-CoA, or an enolic inter mediate sharing a proton with His-274 in a ''low-barrier'' hydrogen bo nd have been suggested. Similar problems of intermediate instability h ave been noted in other enzymic carbon acid deprotonation reactions. Q uantum mechanical/molecular mechanical calculations of the pathway of acetyl-CoA enolization within citrate synthase support the identificat ion of Asp-375 as the catalytic base. His-274, the proposed general ac id, is found to be neutral. The acetyl-CoA enolate is more stable at t he active site than the enol, and is stabilized by hydrogen bonds from His-274 and a water molecule. The conditions for formation of a low-b arrier hydrogen bond do not appear to be met, and the calculated hydro gen bond stabilization in the reaction is less than the gas-phase ener gy, due to interactions with Asp-375 at the active site. The enolate c haracter of the intermediate is apparently necessary for the condensat ion reaction to proceed efficiently. Proteins 27:9-25 (C) 1997 Wiley-L iss, Inc.