A mechanistic proposal for the hydrolytic action of carboxypeptidase A in which the free carboxylate group of glutamate 270 in the enzyme attacks the carbonyl group of the substrate, possibly with electrophilic assistance by the active site zinc, yielding an anhydride intermediate which subsequently breaks down, is being tested in our laboratory using carboxylic ester and thiolester substrates. The experimental probes being employed include the elucidation of structure- reactivity relationships for carboxypeptidase-catalyzed ester hydrolysis, the measurement of kinetic solvent isotope effects for the reactions of various series of specific and nonspecific substrates, and the determination of the effects of changes in the nature of the active site metal ion on the reactivity behavior of the thiolester substrates.