Unveiling the Catalytic Mechanism of a Processive Metalloaminopeptidase

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.