Unveiling the Catalytic Mechanism of a Processive Metalloaminopeptidase

Abstract

Intracellular leucine aminopeptidases (PepA) are metalloproteases from the family M17. These enzymes catalyze peptide bond cleavage, removing N-terminal residues from peptide and protein substrates, with consequences for protein homeostasis and quality control. While general mechanistic studies using model substrates have been conducted on PepA enzymes from various organisms, specific information about their substrate preferences and promiscuity, choice of metal, activation mechanisms, and the steps that limit steady-state turnover remain unexplored. Here, we dissected the catalytic and chemical mechanisms of PaPepA: a leucine aminopeptidase from Pseudomonas aeruginosa. Cleavage assays using peptides and small-molecule substrate mimics allowed us to propose a mechanism for catalysis. Steady-state and pre-steady-state kinetics, pH rate profiles, solvent kinetic isotope effects, and biophysical techniques were used to evaluate metal binding and activation. This revealed that metal binding to a tight affinity site is insufficient for enzyme activity; binding to a weaker affinity site is essential for catalysis. Progress curves for peptide hydrolysis and crystal structures of free and inhibitor-bound PaPepA revealed that PaPepA cleaves peptide substrates in a processive manner. We propose three distinct modes for activity regulation: tight packing of PaPepA in a hexameric assembly controls substrate length and reaction processivity; the product leucine acts as an inhibitor, and the high concentration of metal ions required for activation limits catalytic turnover. Our work uncovers catalysis by a metalloaminopeptidase, revealing the intricacies of metal activation and substrate selection. This will pave the way for a deeper understanding of metalloenzymes and processive peptidases/proteases.

Publication DOI: https://doi.org/10.1021/acs.biochem.3c00420
Divisions: College of Engineering & Physical Sciences > School of Computer Science and Digital Technologies > Software Engineering & Cybersecurity
College of Engineering & Physical Sciences > School of Computer Science and Digital Technologies
Funding Information: C.M.C. is funded by the Wellcome Trust (210486/Z/18/Z and [204821/Z/16/Z] to the University of St Andrews). M.C.S. is funded by a PhD studentship from the University of St Andrews. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1).
Additional Information: Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under CC-BY 4.0. Funding: C.M.C. is funded by the Wellcome Trust (210486/Z/18/Z and [204821/Z/16/Z] to the University of St Andrews). M.C.S. is funded by a PhD studentship from the University of St Andrews. B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1)
Uncontrolled Keywords: Catalysis,Hydrolysis,Kinetics,Leucine/metabolism,Leucyl Aminopeptidase/chemistry,Metals/metabolism,Peptides/metabolism,Substrate Specificity,Biochemistry
Publication ISSN: 1520-4995
Last Modified: 14 Jun 2024 07:33
Date Deposited: 13 Nov 2023 11:38
Full Text Link:
Related URLs: https://pubs.ac ... biochem.3c00420 (Publisher URL)
http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2023-11-21
Published Online Date: 2023-11-04
Accepted Date: 2023-10-11
Authors: Simpson, Martha Clementine
Harding, Christopher John
Czekster, Ricardo Melo (ORCID Profile 0000-0002-6636-4398)
Remmel, Laura
Bode, Bela E.
Czekster, Clarissa Melo

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