The plot of kcat/Km against pH for the Bacillus cereus569/H β-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 µM. 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 β-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.

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