The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

Abstract

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C 6H 5CONHCH 2CONHCH 2CO 2CRR 1CO 2H ( 2 ) and seven esters of N-pivaloylglycine ((CH 3) 3CCONHCH 2CRR 1CO 2H ( 3 ) by bovine pancreatic carboxypeptidase A (Peptidyl- l-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25°C and ionic strength 0.5. All N-hippurylglycine esters ( 2 : R  H, R 1  H, C 2H 5, 4-ClC 6H 4, C 6H 5CH 2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics ( 3 : RH, R 1H, C 2H 5, C 6H 5), substrate activation ( 3 : RH, R 1  4-ClC 6H 4; R  R 1  CH 3) or substrate inhibition ( 3 : R  H, R 1  (CH 3) 2CHCH 2, C 6H 5CH 2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters ( 1 ). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 , 2 and 3 and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.