A detailed kinetic study of Mox-1, a plasmid-encoded class C β-lactamase

Abstract

Surveys of β-lactamases in different parts of the world show an important increase in class C β-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C β-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38 246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k_cat/K_m values >2.5 ×10⁶ M⁻¹ s⁻¹). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K_m values >200 µM). Clavulanic acid had no inhibitory effect on Mox-1 (K_m=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K_i=2.85 µM).