Acetyl-CoA enolization in citrate synthase: A quantum mechanical/molecular mechanical (QM/MM) study

Abstract

Citrate synthase forms citrate by deprotonation of acetyl-CoA followed by nucleophilic attack of this substrate on oxaloacetate, and subsequent hydrolysis. The rapid reaction rate is puzzling because of the instability of the postulated nucleophilic intermediate, the enolate of acetyl-CoA. As alternatives, the enol of acetyl-CoA, or an enolic intermediate sharing a proton with His-274 in a "low-barrier" hydrogen bond have been suggested. Similar problems of intermediate instability have been noted in other enzymic carbon acid deprotonation reactions. Quantum mechanical/molecular mechanical calculations of the pathway of acetyl-CoA enolization within citrate synthase support the identification of Asp-375 as the catalytic base. His-274, the proposed general acid, is found to be neutral. The acetyl-CoA enolate is more stable at the 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-barrier hydrogen bond do not appear to be met, and the calculated hydrogen bond stabilization in the reaction is less than the gas-phase energy, due to interactions with Asp-375 at the active site. The enolate character of the intermediate is apparently necessary for the condensation reaction to proceed efficiently.

Ab initio QM/MM modelling of acetyl-CoA deprotonation in the enzyme citrate synthase

“…Reaction mechanism of citrate synthase, indicating the substrate (keto) and possible intermediate (enolate and enol) forms of acetyl-CoA. bound to chicken citrate synthase produced by semiempirical QM/MM calculations [20]. The simulation system was generally as described for the earlier calculations [20], with some differences as noted below.…”

“…bound to chicken citrate synthase produced by semiempirical QM/MM calculations [20]. The simulation system was generally as described for the earlier calculations [20], with some differences as noted below. Briefly, it consisted of a 17 Å radius sphere, containing 1717 atoms, around the active site taken from a high-resolution crystal structure of chicken citrate synthase complexed with acetyl-CoA and the inhibitor Rmalate [29].…”

“…The calculations build on earlier semiempirical QM/MM studies [20], and on QM calculations on proton transfer in gas-phase models [43]. The results provide no support for the proposal that a low-barrier hydrogen bond stabilizes the intermediate in citrate synthase.…”

“…We have carried out ab initio QM/MM calculations on the reaction catalysed by citrate synthase to test conclusions drawn from earlier studies which used a semiempirical QM treatment [20], and to study the performance of the ab initio QM/MM method. Citrate synthase catalyses the formation of citrate from acetyl-coenzyme A (acetyl-CoA) and oxaloacetate in the citric acid cycle [21,22].…”

“…It has been uncertain whether the nucleophilic intermediate is the enolate or enol of acetylCoA (Scheme 1) [33], or alternatively an 'enolic' form, sharing a proton with His274 in a 'low-barrier' hydrogen bond [34,35]. This is an important question, relating directly to the mechanisms used by this and other enzymes to stabilize reaction intermediates and attain rapid reaction rates [20]. It has been proposed that the enolate is too unstable to be consistent with the observed rate of reaction, unless it is significantly stabilized by the enzyme [36].…”