CATALYTIC HYDROLYSIS OF ACYLCYANAMIDE SUBSTRATES BY CARBOXYPEPTIDASE-A DEFINES THE STEREOCHEMISTRY OF CARBONYL HYDRATION AT THE ENZYME ACTIVE-SITE

New peptide and ester substrate analogues for carboxypeptidase A have been prepared which differ by the possession of an acylcyanamide residue of comparable acidity in place of the C-terminal carboxylic acid group that is normally required by the enzyme. The surrogates are cleaved respectively 2 x 10(5) and 10(4) times more slowly than corresponding regular substrates (k(cat)/K-m). The suggested interpretation is that the carboxylate functionality of normal substrates, in addition to conferring specificity, participates in the enzymic reaction mechanism as a general base, and that this becomes disallowed in the new analogues by specific-binding interactions which induce the cyano substituent to interfere with hydration of the scissile linkage. That reasoning controverts widely accepted ideas regarding the stereochemical course of H2O addition to the enzymic Zn2+-activated peptide linkage as well as the role of carboxypeptidase active site residue Glu 270 in the catalytic mechanism. (C) 1994 Academic Press, Inc.