FLUXIONATE LEWIS ACIDITY OF THE ZN-2+ ION IN CARBOXYPEPTIDASE-A

Competitive inhibition constants K(i) for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of K(i) shows that binding is maximal between two pK(a) values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pK(a) of a Zn2+-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.