Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase
MetadataShow full item record
Prolyl oligopeptidase B from Galerina marginata (GmPOPB) has recently been discovered as a peptidase capable of breaking and forming peptide bonds to yield a cyclic peptide. Despite the relevance of prolyl oligopeptidases in human biology and disease, a kinetic analysis pinpointing rate-limiting steps for a member of this enzyme family is not available. Macrocyclase enzymes are currently exploited to produce cyclic peptides with potential therapeutic applications. Cyclic peptides are promising drug-like molecules due to their stability and conformational rigidity. Here we describe an in-depth kinetic characterization of a prolyl oligopeptidase acting as a macrocyclase enzyme. By combining steady-state and pre-steady-state kinetics, we put forward a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where cyclic peptide departs first followed by the peptide tail. Dissociation of the peptide tail is slow and significantly contributes to the turnover rate. Furthermore, trapping of the enzyme by the peptide tail becomes significant beyond initial-rate conditions. The presence of a burst of product formation and a large viscosity effect further support the rate-limiting nature of a physical step occurring after macrocyclization. This is the first detailed description of the kinetic sequence of a macrocyclase enzyme from this class. GmPOPB is amongst the fastest macrocyclases described to date, and this work is a necessary step towards designing broad specificity efficient macrocyclases.
Czekster , C M & Naismith , J H 2017 , ' Kinetic landscape of a peptide-bond-forming prolyl oligopeptidase ' Biochemistry , vol 56 , no. 15 , pp. 2086-2095 . DOI: 10.1021/acs.biochem.7b00012
Copyright © 2017 American Chemical Society. This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
We thank Dr. Rafael Guimaraes da Silva for helpful discussions on enzyme kinetics. We also thank Professor David Lilley, Dr. Alasdair Freeman and Dr. Anne-Cecile Declais at the University of Dundee for training and usage of their QFM-4000 quenched-flow apparatus.
Items in the St Andrews Research Repository are protected by copyright, with all rights reserved, unless otherwise indicated.