Author:
Dubée Vincent,Triboulet Sébastien,Mainardi Jean-Luc,Ethève-Quelquejeu Mélanie,Gutmann Laurent,Marie Arul,Dubost Lionel,Hugonnet Jean-Emmanuel,Arthur Michel
Abstract
ABSTRACTThe structure ofMycobacterium tuberculosispeptidoglycan is atypical since it contains a majority of 3→3 cross-links synthesized byl,d-transpeptidases that replace 4→3 cross-links formed by thed,d-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate thesel,d-transpeptidases, and meropenem combined with clavulanic acid is bactericidal against extensively drug-resistantM. tuberculosis. Here, we used mass spectrometry and stopped-flow fluorimetry to investigate the kinetics and mechanisms of inactivation of the prototypicM. tuberculosisl,d-transpeptidase LdtMt1by carbapenems (meropenem, doripenem, imipenem, and ertapenem) and cephalosporins (cefotaxime, cephalothin, and ceftriaxone). Inactivation proceeded through noncovalent drug binding and acylation of the catalytic Cys of LdtMt1, which was eventually followed by hydrolysis of the resulting acylenzyme. Meropenem rapidly inhibited LdtMt1, with a binding rate constant of 0.08 μM−1min−1. The enzyme was unable to recover from this initial binding step since the dissociation rate constant of the noncovalent complex was low (<0.1 min−1) in comparison to the acylation rate constant (3.1 min−1). The covalent adduct resulting from enzyme acylation was stable, with a hydrolysis rate constant of 1.0 × 10−3min−1. Variations in the carbapenem side chains affected both the binding and acylation steps, ertapenem being the most efficient LdtMt1inactivator. Cephalosporins also formed covalent adducts with LdtMt1, although the acylation reaction was 7- to 1,000-fold slower and led to elimination of one of the drug side chains. Comparison of kinetic constants for drug binding, acylation, and acylenzyme hydrolysis indicates that carbapenems and cephems can both be tailored to optimize peptidoglycan synthesis inhibition inM. tuberculosis.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Pharmacology (medical),Pharmacology
Cited by
136 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献