Interaction of carbapenems and β-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238C mutant.

Abstract

The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238C mutant with respect to carbapenems and various β-lactamase inhibitors. A CTX-M-15G238C laboratory mutant was generated by site-directed mutagenesis from CTX-M-15 enzyme by replacing glycine 238 with cysteine. Thiol titration and p-chloromercuribenzoate (PCMB) inactivation assays were used to ascertain the presence of a disulfide bridge in the active site of CTX-M-15G238C. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15G238C enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. In CTX-M-15G238C mutant, the two cysteines (C69 and C238) located in the enzyme active site were unable to form a disulfide bridge. CTX-M-15 and thermostable CTX-M-15G238C were used to study the kinetic interaction with carbapenems, which behaved as poor substrates for both enzymes. Meropenem and ertapenem acted as transient inactivators for CTX-M-15 and CTX-M-15G238C, and for these compounds the variation of kobs versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k+3=0) for CTX-M-15G238C. In any case, the k+2/K values for CTX-M-15G238C were higher than those for CTX-M-15. Compared with CTX-M-15, CTX-M-15G238C mutant appears to have a more favourable conformation for carbapenem acylation and higher activity against cefotaxime, which could be due to the presence of free -SH groups in the enzyme active site.