Carboxypeptidase inhibitor from potatoes. Interaction with derivatives of carboxypeptidase A

Abstract

The mechanism of action of a carboxypeptidase inhibitor from potatoes was probed by studying its interaction with derivatives of carboxypeptidase A containing modified residues at the active site. Arsanilazocarboxypeptidase A, a derivative containing a chromophore attached to tyrosine 248, exhibits a circular dichroic spectrum which is sensitive to the presence of ligands at the active site. Since the spectral change attending binding of the carboxypeptidase inhibitor to arsanilazocarboxypeptidase A is similar to that produced by small substrates and inhibitors, the enzyme-inhibitor interaction also involves the enzyme active site. Catalytic activity is not required for inhibitor binding. Complexes of the inhibitor with apocarboxypeptidase A and carboxypeptidase A which was inactivated by treatment with the affinity label, N-bromoacetyl-N-methyl-L-phenylalanine, are demonstrated by gel filtration experiments. Competitive binding studies reveal that the latter derivative, in which the binding pocket is presumably blocked by reagent, binds inhibitor nearly as strongly as does the native enzyme, the differences in free energy of association being only 0.4 kcal/mol of a total binding energy of -11 kcal/mol. A model is proposed to account for both the tight binding of inhibitor to the N-bromoacetyl-N-methyl-L-phenylalanine derivative and the involvement of the active site of arsanilazocarboxypeptidase A. The inhibitor may fit into a shallow depression at the active site of the enzyme but does not penetrate into the binding pocket.