title: The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase.
abstract: The plot of kcat/Km against pH for the Bacillus cereus 569/H beta-lactamase class B catalysed hydrolysis of benzylpenicillin and cephalosporin indicates that there are three catalytically important groups, two of pKa 5.6+/-0.2 and one of pKa 9.5+/-0.2. Below pH 5 there is an inverse second-order dependence of reactivity upon hydrogen ion concentration, indicative of the requirement of two basic residues for catalysis. These are assigned to zinc(II)-bound water and Asp-90, both with a pKa of 5.6+/-0.2. A thiol, N-(2'-mercaptoethyl)-2-phenylacetamide, is an inhibitor of the class B enzyme with a Ki of 70 microM. The pH-dependence of Ki shows similar pH inflections to those observed in the catalysed hydrolysis of substrates. The pH-independence of Ki between pH 6 and 9 indicates that the pKa of zinc(II)-bound water must be 5.6 and not the higher pKa of 9.5. The kinetic solvent isotope effect on kcat/Km is 1.3+/-0.5 and that on kcat is 1.5. There is no effect on reactivity by either added zinc(II) or methanol. The possible mechanisms of action for the class B beta-lactamase are discussed, and it is concluded that zinc(II) acts as a Lewis acid to stabilize the dianionic form of the tetrahedral intermediate and to provide a hydroxide-ion bound nucleophile, whereas the carboxylate anion of Asp-90 acts as a general base to form the dianion and also, presumably, as a general acid catalyst facilitating C-N bond fission.
authors.affiliation: Department of Chemical and Biological Sciences, The University of Huddersfield, Queensgate, Huddersfield, Yorkshire HD1 3DH, UK.
authors.forename: S
authors.initials: S
authors.lastname: Bounaga
authors.forename: A P
authors.initials: AP
authors.lastname: Laws
authors.forename: M
authors.initials: M
authors.lastname: Galleni
authors.forename: M I
authors.initials: MI
authors.lastname: Page
mesh_terms: D000081:Acetamides / Q000494:pharmacology*; D000083:Acetanilides; D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D002509:Cephaloridine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D000432:Methanol / Q000494:pharmacology; D015394:Molecular Structure; D009994:Osmolar Concentration; D010400:Penicillin G / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000378:metabolism*
chemical_list: D000081:Acetamides; D000083:Acetanilides; C114911:N-(2'-mercaptoethyl)-2-phenylacetamide; D013438:Sulfhydryl Compounds; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases; D015032:Zinc; D002509:Cephaloridine; D010400:Penicillin G; D000432:Methanol