Kinetic studies on the chymotrypsin A α-catalyzed hydrolysis of a series of N-acetyl- l-phenylalanyl peptides

Abstract

A series of N-acetyl- l-phenylalanyl peptides of general formula Ac-Phe-(Gly) n -NH 2 ( n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH 2, AcPheAlaGlyNH 2, and AcPheGlyAlaNH 2. Values of k cat and K m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.