The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-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.

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…This moiety is asymmetrically positioned with respect to the two metal ions, lying closer (1.9 -2.1 Å) to the metal ion in the 3H site than to the Zn(II) ion in the DCH or DHH sites (2.1-3.1 Å) (13,14,28). The shorter bond length in the former case is consistent with this ligand being a hydroxide and with the idea that the 3H site is responsible for lowering the pK a of a water molecule, thus being responsible for nucleophile activation (57). The role of CϭO polarization by this same zinc ion is not supported by QM/MM calculations and by different docking studies, which reveal that the ␤-lactam bond may not directly bind to the metal ion (60,61).…”

“…This fact highlights its amazing chemical versatility. In the case of M␤Ls, the Zn(II) ion is able to contribute to ␤-lactam hydrolysis by 1) lowering the pK a of a bound water molecule, which may act as a nucleophile, providing a high local concentration of hydroxide ions at neutral pH (37,57); 2) acting as a Lewis acid, polarizing the CϭO bond and therefore augmenting the electrophilic nature of the carbonyl carbon (57); and 3) stabilizing a negative charge in the bridging nitrogen of the lactam moiety, after C-N bond cleavage (16,17,24,36,58,59). These three roles have been invoked for the two metal binding sites in M␤Ls, but it is not clear yet which of them are essential for catalysis.…”

Substrate-activated Zinc Binding of Metallo-β-lactamases

“…Subclass B3 enzymes exhibit a broad spectrum activity profile with a putative preference for cephalosporins (5). The question as to whether zinc-␤-lactamases are active as the mono-or the dizinc enzyme has been controversially discussed, and two alternative mechanisms for both enzyme states have been proposed (6,7).…”

Virtual Screening of Penicillin-derived Inhibitors for the Metallo-β-lactamase from Bacillus cereus

“…Particularly, compounds 5a and 5b had the ability to selectively and actively inhibit the MβL from Bacillus cereus (BcII), whose mechanistic and structural information has been elucidated in previous MβL studies. 25 Compound 5b had the best BcII inhibitory activity among the four suggested compounds, with an IC50 value of 1.7 µM 23 (Table 1).…”