Kinetic Characterization of Hydrolysis of Nitrocefin, Cefoxitin, and Meropenem by β-Lactamase fromMycobacterium tuberculosis

Abstract

The constitutively expressed, chromosomally-encoded β-lactamase (BlaC) is the enzyme responsible for the intrinsic resistance to β-lactam antibiotics in Mycobacterium tuberculosis. Previous studies from this laboratory have shown that the enzyme exhibits an extended-spectrum phenotype, with very high levels of penicillinase and cephalosporinase activity, as well as weak carbapenemase activity (1). In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximum velocity depends on the ionization state of two groups; a general base exhibiting a pK value of 4.5 and a general acid exhibiting a pK value of 7.8. Having defined a region where the kinetic parameters are pH-independent (pH 6.5), we determined solvent kinetic isotope effects (SKIEs) for three substrates whose kcat values differ by 4.5 orders of magnitude. Nitrocefin is a highly activated, chromogenic cephalosporin derivative that exhibits steady-state solvent kinetic isotope effects of 1.4 on both V and V/K. Cefoxitin is a slower cephalosporin derivative that exhibits a large SKIE on V of 3.9, but a small SKIE of 1.8 on V/K in steady-state experiments. Pre-steady-state, stopped-flow experiments with cefoxitin revealed a burst of β-lactam ring opening with associated SKIE values of 1.6 on the acylation step and 3.4 on the deacylation step. Meropenem is an extremely slow substrate for BlaC, and exhibits burst kinetics in the steady-state experiments. SKIE determinations with meropenem revealed large SKIEs on both the acylation and deacylation step of 3.8 and 4.0, respectively. Proton inventories in all cases were linear, indicating the participation of a single solvent-derived proton in the chemical step responsible for the SKIE. The rate limiting steps for β-lactam hydrolysis of these substrates are analyzed, and the chemical step(s) responsible for the observed SKIE are discussed.

Carbapenems and Rifampin Exhibit Synergy against Mycobacterium tuberculosis and Mycobacterium abscessus

“…(Carba)penems are known to be either slow substrates or inhibitors of BlaC, a ␤-lactamase of M. tuberculosis (2,5,(27)(28)(29)(30). A 2-to 4-fold reduction in MIC 90 was seen for all (carba)penems except faropenem when supplemented with 5 g/ml clavulanic acid.…”

Mechanisms of proton relay and product release by Class A β‐lactamase at ultrahigh resolution

“…7A) [37], and underscores the evolutionary connection between PBPs and Class A b-lactamases. The introduction of Glu166 in the active site of Class A b-lactamase increases the pKa of Lys73 from~6 to 8-8.5, making this lysine more likely to be positively charged at physiological pH and hindering its role as general base [44,45,[47][48][49]. A positively charged Lys73 was indeed observed in WT CTX-M-14 by ultrahigh resolution X-ray crystallography [19,37].…”

Molecular Determinants for Protein Stabilization by Insertional Fusion to a Thermophilic Host Protein

“…The rate of CENTA hydrolysis was similarly measured at 37 °C. Changes in the molecular extinction coefficient (Δɛ) of CENTA and nitrocefin upon complete hydrolysis were reported to be 6400 m −1 cm −1 at 405 nm and 20 500 m −1 cm −1 at 486 nm, respectively, and were used for evaluation of hydrolysis rates. Michaelis–Menten parameters were then determined from the initial rate of reaction at various substrate concentrations by using Eadie–Hofstee plots.…”