Ability of single-site mutants of citrate synthase to catalyze proton transfer from the methyl group of dethiaacetyl-coenzyme A, a non-thioester substrate analog

The optimal induction conditions and solubility levels of each mutant porcine CS genes expressed in Escherichia coli were analyzed to determine the mechanisms of citrate synthase-catalyzed proton transfer. Citrate synthase activity was sensitive to steric factors, differences in the bond length of the C-S-C unit of the natural thioester substrate and the C-C-C of the ketone analog. Furthermore, citrate synthase-catalyzed proton transfer was also dependent on the activation of the oxaloacetate substrate through the polarization of the carbonyl bonds.