Kinetic Landscape of a Peptide Bond-Forming Prolyl Oligopeptidase.
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 druglike molecules because of 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 propose a kinetic sequence in which a step after macrocyclization limits steady-state turnover. Additionally, product release is ordered, where the 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 among the fastest macrocyclases described to date, and this work is a necessary step toward designing broad-specificity efficient macrocyclases.
School of Chemistry, Biomedical Sciences Research Complex, University of St Andrews , North Haugh, St Andrews K16 9ST, U.K.
Clarissa M
0000-0002-7163-4057
CM
Czekster
School of Chemistry, Biomedical Sciences Research Complex, University of St Andrews , North Haugh, St Andrews K16 9ST, U.K.|Biotherapy Centre, Sichuan University , Chengdu, China.
James H
0000-0001-6744-5061
JH
Naismith
D002151:Calorimetry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry*; D000085822:Prolyl Oligopeptidases; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013696:Temperature; D014783:Viscosity
D010455:Peptides; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases