Objectives The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238Cmutant with respect to carbapenems and various β-lactamase inhibitors. Methods A CTX-M-15G238Claboratory 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-15G238Cenzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. Results In CTX-M-15G238Cmutant, 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-15G238Cwere 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 kobsversus 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-15G238Cwere higher than those for CTX-M-15. Conclusions Compared with CTX-M-15, CTX-M-15G238Cmutant 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.

Interaction of carbapenems and β-lactamase inhibitors towards CTX-M-15 and CTX-M-15G238Cmutant

Sabatini, Alessia;Brisdelli, Fabrizia;Celenza, Giuseppe;Marcoccia, Francesca;Colapietro, Martina;Piccirilli, Alessandra;Amicosante, Gianfranco;Perilli, Mariagrazia
2017-01-01

Abstract

Objectives The aim of this study was to evaluate the role of residue 238 in CTX-M-15 and CTX-M-15G238Cmutant with respect to carbapenems and various β-lactamase inhibitors. Methods A CTX-M-15G238Claboratory 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-15G238Cenzymes by analysing either the complete hydrolysis time courses or under initial rate conditions. Results In CTX-M-15G238Cmutant, 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-15G238Cwere 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 kobsversus 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-15G238Cwere higher than those for CTX-M-15. Conclusions Compared with CTX-M-15, CTX-M-15G238Cmutant 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.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11697/121273
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