Binding of ligands to the active site of carboxypeptidase A.

Abstract

The detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analog (-)-2-benzyl-3-p-methoxybenzoylpropionate and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1) are compared. In the potato inhibitor, cleavage of the COOH-terminal Gly-39 leaves a new carboxylate anion of Val-38 having 1 oxygen on Zn and the other as a receptor of a H-bond from Tyr-248 of carboxypeptidase. Tyr-248 also receives a H-bond from the amide proton of the originally penultimate peptide bond between Tyr-37 and Val-38. This H-bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (Tyr-198, Phe-279 and Arg-71) and preceding entry into the catalytic site S1. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S''1, as a competitive inhibitor for esters (for which entry into S''1 precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S''1 is rate limiting). These results, including the binding mode of the ester analog, are consistent with the original proposal from X-ray studies that both esters and peptides are cleaved with the carboxy terminus at S''1, although not necessarily by the same chemical steps.