Citrate synthase stabilizes the enethiolate of acetyldithio-coenzyme A

Abstract

Citrate synthase catalyzes the slow condensation of acetyldithio-CoA [Ac(= S)CoA] with oxalacetate to form thiocitrate [Wlassics, I.D., Stille, C., & Anderson, V.E. (1988) Biochim. Biophys. Acta 952, 269]. During the transient approach to steady state an observable amount of the dithioester absorbance disappears. The amplitude of the decrease in absorbance corresponds to 0.32, 0.03, and 0.02 enzyme equiv at pH 8.3, 7.5, and 6.6, respectively. The difference spectra from before and after the transient exhibit the dithioester lambda max at 306 nm. Acid quenching of a stiochiometric reaction between Ac(= S)CoA and citrate synthase following the transient quantitatively regenerates Ac(= S)CoA, indicating carbon-carbon bond formation had not yet occurred. The apparent first-order rate constant of the transient is independent of Ac(= S)CoA concentration and increases with decreasing pH, being 0.007, 0.016, and 0.04 s-1 at pH 8.3, 7.5, and 6.6, respectively. 2-Fluoroacetyldithio-CoA is a better inhibitor of citrate synthase, Ki = 300 nM, and substrate, Vmax = 2 X 10(-3) s-1, than Ac(= S)CoA. 1H NMR experiments indicate that citrate synthase catalyzes the exchange of the alpha-hydrogens of Ac(= S)CoA with turnover numbers of 0.13 and 0.54 s-1 at pD 7.9 and 7.2, respectively. Analysis of the proton and deuterium decoupled 13C NMR spectra of [2-13C]Ac(= S)CoA that has exchanged 37% of the alpha-hydrogens in the presence of citrate synthase indicates that the relative proportions of CH3, CH2D, CHD2, and CD3 were 0.29, 0.39, 0.25, and 0.07, respectively. This statistical distribution indicates each exchange event is independent. The data indicate that citrate synthase stabilizes the ionized form of Ac(= S)CoA by 5 kcal/mol relative to the un-ionized form, that the ionized dithioester is on the reaction pathway, and that below pH 8.3 the slow carbon-carbon bond forming reaction is responsible for the 10(6) decrease in Vmax caused by substituting sulfur for oxygen in the thioester carbonyl.

An atomic-resolution mechanism of 3-hydroxy-3-methylglutaryl–CoA synthase

“…In pingpong fashion the second substrate, acetoacetyl-CoA (AcAc-CoA), enters after the release of CoASH, thereby setting up the more challenging condensation reaction. As in the case here, features of enzymes that proceed through a carbon enolate intermediate or in a concerted fashion with carbanion character have long been a mechanistic question for a variety of systems (8)(9)(10)(11). In the case of HMG-CoA synthase, Theisen et al (7) had the good fortune to cryogenically trap protein crystals that had, to a varying extent, two of these mechanistically revealing states.…”

Active Site Probes for Yeast OMP Decarboxylase: Inhibition Constants of UMP and Thio-Substituted UMP Analogues and Greatly Reduced Activity toward CMP-6-Carboxylate

“…In acetyldithio-CoA, S has replaced O at the carbonyl group in acetyl-CoA that undergoes polarization by citrate synthase in the transition state of citryl-CoA formation (21). In the reaction with acetyl-CoA, polarization occurs upon interaction of the carbonyl O with an enzyme histidyl side chain, resulting in a strong hydrogen bond proposed to occur only in the transition state (22).…”

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

“…In citrate synthase, debate has centered on the identity of the nucleophilic intermediate, which is believed to be formed by deprotonation of acetyl-CoA. This deprotonation is probably rate-limiting [22,62], and is not concerted with the subsequent nucleophilic attack on oxaloacetate, based on experimental results for the similar malate synthase reaction [24], and for a substrate analog in citrate synthase [23]; model calculations also show a barrier to the condensation reaction [6]. The puzzle has then been how the enzyme is able to rapidly deprotonate a weakly acidic thioester using only amino acid sidechains.…”

“…Citrate synthase catalyses the formation of citrate from acetyl-coenzyme A (acetyl-CoA) and oxaloacetate in the citric acid cycle [21,22]. The reaction proceeds via deprotonation of acetyl-CoA to form a nucleophilic intermediate [6,23,24], which subsequently attacks the carbonyl carbon of oxaloacetate, which is polarized at the active site [25,26]. Hydrolysis of the thioester bond then allows release of the products.…”