The Role of the Nonconserved Residues at Position 167 of Class A β-Lactamases in Susceptibility to Mechanism-Based Inhibitors

Abstract

ABSTRACT

Differences in specificities between the class A β-lactamases for both substrate and inhibitors are known. The role of the nonconserved amino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both the hydrolysis of β-lactam substrates and inactivation by mechanism-based inhibitors, was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H β-lactamase I to replace thr-167 with the corresponding Staphylococcus aureus PC1 residue Ile. Kinetic data obtained from the purified Thr-167-Ile B. cereus 569/H β-lactamase was compared to that obtained from the wild-type B. cereus and S. aureus enzymes and indicated that the replacement had little effect on the Michaelis parameters for the hydrolysis of S- and A-type penicillins. However, the Thr-167-Ile enzymes became more S. aureus PC1-like in its response to the mechanism-based inhibitors clavulanic acid and 6-β-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone. A model for the role of this nonconserved residue at position 167 in the mechanism of inactivation by mechanism-based inhibitors is proposed.