pH, inhibitor, and substrate specificity studies on Escherichia coli penicillin-binding protein 5

Biochim Biophys Acta. 2002 Jun 3;1597(2):292-300. doi: 10.1016/s0167-4838(02)00311-4.

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins*
  • Carrier Proteins / antagonists & inhibitors*
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism
  • Catalytic Domain
  • Cattle
  • Cell Wall / chemistry
  • Cell Wall / metabolism
  • Enzyme Inhibitors / pharmacology
  • Enzyme Stability
  • Escherichia coli Proteins / antagonists & inhibitors*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism
  • Hexosyltransferases*
  • Hydrogen-Ion Concentration
  • In Vitro Techniques
  • Kinetics
  • Models, Molecular
  • Muramoylpentapeptide Carboxypeptidase / antagonists & inhibitors*
  • Muramoylpentapeptide Carboxypeptidase / chemistry*
  • Muramoylpentapeptide Carboxypeptidase / metabolism
  • Oligopeptides / chemistry
  • Oligopeptides / metabolism
  • Penicillin-Binding Proteins
  • Peptidyl Transferases*
  • Serum Albumin, Bovine
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Carrier Proteins
  • Enzyme Inhibitors
  • Escherichia coli Proteins
  • Oligopeptides
  • Penicillin-Binding Proteins
  • Serum Albumin, Bovine
  • Peptidyl Transferases
  • Hexosyltransferases
  • Muramoylpentapeptide Carboxypeptidase