title: pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5.
abstract: The recent structural determination of Escherichia coli penicillin-binding protein 5 (PBP 5) provides the opportunity for detailed structure-function studies of this enzyme. PBP 5 was investigated in terms of its stability, linear reaction kinetics, acyl-donor substrate specificity, inhibition by a number of active site-directed reagents, and pH profile. PBP 5 demonstrated linear reaction kinetics for up to several hours. Dilution of PBP 5 generally resulted in substantial loss of activity, unless BSA or a BSA derivative was added to the diluting buffer. PBP 5 did not demonstrate a significant preference against a simple set of five alpha- and epsilon-substituted L-Lys-D-Ala-D-Ala derivatives, suggesting that PBP 5 lacks specificity for the cross-linked state of cell wall substrates. Among a number of active site-directed reagents, only some thiol-directed reagents gave substantial inhibition. Notably, serine-directed reagents, organic phosphates, and simple boronic acids were ineffective as inhibitors. PBP 5 was stable over the pH range 4.6-12.3, and the k(cat)/K(m) vs. pH profile for activity against Ac(2)-L-Lys-D-Ala-D-Ala was bell-shaped, with pK(a)s at 8.2 and 11.1. This is the first complete pH profile, including both acidic and basic limbs, for a PBP-catalyzed DD-carboxypeptidase (CPase) reaction. Based on its structure, similarity to Class A beta-lactamases, and results from mutagenesis studies, the acidic and basic limbs of the pH profile of PBP 5 are assigned to Lys-47 and Lys-213, respectively. This assignment supports a role for Lys-47 as the general base for acylation and deacylation reactions.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110, USA.
authors.forename: Miglena E
authors.initials: ME
authors.lastname: Stefanova
authors.forename: Christopher
authors.initials: C
authors.lastname: Davies
authors.forename: Robert A
authors.initials: RA
authors.lastname: Nicholas
authors.forename: William G
authors.initials: WG
authors.lastname: Gutheil
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002417:Cattle; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D006602:Hexosyltransferases; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D002267:Muramoylpentapeptide Carboxypeptidase / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D046915:Penicillin-Binding Proteins; D010458:Peptidyl Transferases; D012710:Serum Albumin, Bovine; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D009842:Oligopeptides; D046915:Penicillin-Binding Proteins; D012710:Serum Albumin, Bovine; D010458:Peptidyl Transferases; D006602:Hexosyltransferases; D002267:Muramoylpentapeptide Carboxypeptidase