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

Objectives: The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15 mutant with respect to carbapenems and various β-lactamase inhibitors.

Methods: A CTX-M-15 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-15. Kinetic parameters were determined both for CTX-M-15 and CTX-M-15 enzymes by analysing either the complete hydrolysis time courses or under initial rate conditions.

Results: In CTX-M-15 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-15 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-15, and for these compounds the variation of k versus the inactivator concentration was linear. Imipenem behaved as a transient inactivator for CTX-M-15 and as an inactivator (with k=0) for CTX-M-15. In any case, the k/K values for CTX-M-15 were higher than those for CTX-M-15.

Conclusions: Compared with CTX-M-15, CTX-M-15 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.