An investigation of the active site of Aeromonas aminopeptidase

Abstract

The mechanism of the hydrolysis of L-leucyl-p-nitroanilide by Aeromonas aminopeptidase has been investigated by a combination of pH-dependence, competitive inhibition, and chemical modification studies. The pH-dependence of the buffer-independent value of K[subscript m] indicates that binding of the substrate to the enzyme requires the free-base form of a group (presumably the substrate α-amino group) ionizing near pH 7.5, and the undissociated form of two or three groups ionizing in the vicinity of pH 10.0. The pH-dependence of the catalytic rate constant, k[subscript cat], indicates that the free-base form of an enzyme functional group with pK[subscript a] near 5.6 is required for hydrolysis of bound substrate. Two models are proposed to explain the pH-dependence of k[subscript cat] and K[subscript m]; one of these models yields estimates of 21.6 sec⁻¹ and 7.5 x 10⁶ M⁻¹sec⁻¹, respectively, for the rate constants for dissociation and recombination of enzyme and substrate. ...