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 catalytic strategies of enzymes (such as citrate synthase) whose reactions require the abstraction of the α-proton of a carbon acid remain elusive. Citrate synthase readily catalyzes solvent proton exchange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloacetate. Because no further reaction occurs with this analog, it provides a uniquely simple probe of the roles of active site interactions on carbon acid proton transfer catalysis. In view of the high reactivity of the analog for proton transfer to the active site base, its failure to further condense with oxaloacetate to form a sulfur-less analog of citryl-coenzyme A was unexpected, although we offer several possible explanations. We have measured the rate constants for exchange, kexch, at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues. Comparisons between the values of kexch are straightforward i...