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 r eactions require the abstraction of the alpha-proton of a carbon acid remain elusive. Citrate synthase readily catalyzes solvent proton exch ange of the methyl protons of dethiaacetyl-coenzyme A, a sulfur-less, ketone analog of acetyl-coenzyme A, in its ternary complex with oxaloa cetate. Because no further reaction occurs with this analog, it provid es 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 failur e to further condense with oxaloacetate to form a sulfur-less analog o f citryl-coenzyme A was unexpected, although we offer several possible explanations. We have measured the rate constants for exchange, k(exc h), at saturating concentrations of the analog for six citrate synthas e mutants with single changes in active site residues. Comparisons bet ween the values of k(exch) are straightforward in two limits. If the r ate of exchange of the transferred proton with solvent protons is rapi d, then k(exch) equals the forward rate constant for proton transfer, and k(exch) values for different mutants compare directly the rate con stants 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(ex ch) equals the product of the equilibrium constant for proton transfer and the rate constant for exchange of the transferred proton with bul k solvent. If that exchange rate with bulk solution remains constant f or a series of mutant enzymes, then k(exch) values compare the equilib rium constants for proton transfer. The importance of the acetyl-CoA s ite residues, H274 and D375, is confirmed with D375 again implicated a s the active site base. The results with the series of oxaloacetate si te mutants, H320X, strongly suggest that activation of the first subst rate, oxaloacetate, through carbonyl bond polarization, not just oxalo acetate binding in the active site, is required for the enzyme to effi ciently catalyze proton transfer from the methyl group of the second s ubstrate.