Structure and Catalysis of Acylaminoacyl Peptidase

Abstract

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.

Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

“…At last, the results from our simulations, where also the wild type ApAAP open and closed conformations are compared, strongly confirm the mechanism proposed in the literature of a “clamshell-like” conformational rearrangement at the interdomain interface in the proximity of the catalytic site, recently proposed [33]. In this context, the study of correlated motions and the networks of electrostatic interactions at the interface in both open and closed ApAAP conformations on ns time scales, allows to disclose relevant interactions and couple motions which can contribute to the transition between the two forms and which deserve further attention.…”

“…The switch between these two conformational states is likely to be mediated by modification of intermolecular interactions between the two domains and relying on the hinge role of D376 [33]. A similar conformation has been also identified by electron microscopy investigation of a POP enzyme [54].…”

Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms

“…In AAP enzymes, two different mechanisms for substrate screening have been proposed. Dimeric AAP (ApAAP) from Aeropyrum pernix shows dynamic opening and closing of the enzyme that are rather similar to observations in prokaryotic POPs and OPBs (25). In contrast, hexameric PhAAP shows the double-gated entry mechanism.…”

Reciprocal Influence of Protein Domains in the Cold-Adapted Acyl Aminoacyl Peptidase from Sporosarcina psychrophila

“…Experimental studies demonstrated instead that isolated, unclosed β-propellers can be stable and suggested that enzyme activity relies rather on concerted movements of the peptidase and propeller domains [16], [17]. Nowadays a clamshell-like movement has been proposed to describe the substrate-induced conformational changes performed by several prolyl peptidases [18], [19], although analysis of molecular dynamics reveals that such conformational changes can be strikingly different in different enzymes, leading to different selectivity towards the substrate [20].…”