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 alpha-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, k(exch), at saturating concentrations of the analog for six citrate synthase mutants with single changes in active site residues. Comparisons between the values of k(exch) are straightforward in two limits. If the rate of exchange of the transferred proton with solvent protons is rapid, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate constants for proton transfer. If the exchange of the transferred proton with protons in the bulk solution is the slow step and the equilibrium constant for proton transfer is unfavorable (as is likely), then k(exch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bulk solvent. If that exchange rate with bulk solution remains constant for a series of mutant enzymes, then k(exch) values compare the equilibrium constants for proton transfer. The importance of the acetyl-CoA site residues, H274 and D375, is confirmed with D375 again implicated as the active site base. The results with the series of oxaloacetate site mutants, H320X, strongly suggest that activation of the first substrate, oxaloacetate, through carbonyl bond polarization, not just oxaloacetate binding in the active site, is required for the enzyme to efficiently catalyze proton transfer from the methyl group of the second substrate.