THE ROLE OF THE NONCONSERVED RESIDUES AT POSITION-167 OF CLASS-A BETA-LACTAMASES IN SUSCEPTIBILITY TO MECHANISM-BASED INHIBITORS

Differences in specificities between the class A beta-lactamases for b oth substrate and inhibitors are known, The role of the nonconserved a mino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both th e hydrolysis of beta-lactam substrates and inactivation by mechanism-b ased inhibitors, was investigated, Site-directed mutagenesis was perfo rmed on the penPC gene encoding the Bacillus cereus 569/H beta-lactama se I to replace thr-167 with the corresponding Staphylococcus aureus P C1 residue Re. Kinetic data obtained from the purified Thr-167-Ile B, cereus 569/H beta-lactamase was compared to that obtained from the wil d-type B, cereus and S, aureus enzymes and indicated that the replacem ent had little effect on the Michaelis parameters for the hydrolysis o f S- and A-type penicillins, However, the Thr-167-Ile enzymes became m ore S, aureus PC1-like in its response to the mechanism-based inhibito rs clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amidopenicill anic acid sulfone, A model for the role of this nonconserved residue a t position 167 in the mechanism of inactivation by mechanism-based inh ibitors is proposed.