Reversible Inhibition of Carboxypeptidase A. IV. Inhibition of Specific Esterase Activity by Hippuric Acid and Related Species and other Amino Acid Derivatives and a Comparison with Substrate Inhibition

Abstract

The anions of each of the following carboxylic acids exhibit uncompetitive inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by bovine carboxypeptidase A at pH 7.5, 25°, ionic strength 0.2: hippuric acid, p-chloro- and p-nitrohippuric acids, hippurylglycine, carbobenzoxyglycine, phenaceturic acid, N'-(3-phenylpropanoyl)glycine, benzoxyacetic acid, 3-benzoylpropanoic acid, and O-hippuryl-D-mandelic acid. In each case, this uncompetitive inhibition is consistent with the ordered binding of substrate and inhibitor to the enzyme; i.e. the inhibitor binds to E.S but not to the free enzyme. Evidence is presented for the binding site for uncompetitive inhibitors being the same as for inhibitory ester substrate molecules. Comparison of the specificities of uncompetitive inhibitors and esters which display substrate inhibition provides evidence for a critical conformational change which controls the binding of uncompetitive inhibitors and inhibitory substrate molecules.D-Phenylalanine, D-leucine, D-p-nitrophenylalanine, glycyl-L-tyrosine, glycyl-L-phenylalanine, and glycyl-L-leucine are competitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid, whereas the N-chloroacetyl derivatives of L-tyrosine, L-phenylalanine, and L-leucine are noncompetitive inhibitors. For the above D-amino acids, glycyl dipeptides, and N-chloroacetyl amino acids, the phenylalanine derivative in each case is a considerably stronger inhibitor than the corresponding leucine derivative. This preference is similar to that observed for the binding of peptide substrates but the reverse of that observed for ester substrates and simple mono- and dicarboxylate ion inhibitors.The peptide substrates carbobenzoxyglycylglycyl-L-phenylalanine and N-chloroacetyl-L-phenylalanine are noncompetitive inhibitors of the enzymic hydrolysis of O-hippuryl-L-3-phenyllactic acid. This clearly demonstrates the presence of different ester and peptide binding sites in this enzyme, which is consistent with conclusions from recent studies in other laboratories.