Kinetic characterization of hydrolysis of nitrocefin, cefoxitin, and meropenem by β-lactamase from Mycobacterium 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 [Tremblay, L. W., et al. (2008) Biochemistry 47, 5312-5316]. In this report, we have determined the pH dependence of the kinetic parameters, revealing that the maximal 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 5.5 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 steps 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 steps responsible for the observed SKIE are discussed.

Figure 1

pH dependence of BlaC on log k_cat (■) and log k_cat/K_m (□) with (A) nitrocefin and (B) cefoxitin. The lines represent fits to eq 1 and 2. A mixed buffer system was utilized, with 100 mM sodium acetate, 100 mM MES, 100 mM HEPES, and 100 mM TAPS.

Figure 2

Initial velocity pattern for (A) nitrocefin with 2 nM BlaC and (B) cefoxitin with 350 nM BlaC in H₂O (●) and in D₂O (○) with 100 mM MES at pH 6.3.

Figure 3

Proton inventories of (A) nitrocefin with 2 nM BlaC and (B) cefoxitin with 350 nM BlaC measuring the initial velocity with varying % D₂O in 100 mM MES at pH 6.3.

Figure 4

Burst kinetic isotope effects on (A) cefoxitin and (B) meropenem in H₂O (black) and D₂O (gray) with fits of experimental traces to the k₂ (acylation) and k₃ (deacylation) values in Table 1 (red). Cefoxitin studies consisted of 1800 μM cefoxitin mixed with 27 μM BlaC. Meropenem studies consisted of 875 μM meropenem mixed with 13 μM BlaC.

Scheme 1

Structures of benzylpenicillin, nitrocefin, cefoxitin, and meropenem.

Scheme 2

Scheme 3

Proposed catalytic mechanism of BlaC. Activation Ser70 by Lys73 to promote β-lactam ring opening (A), formation of the covalent acyl-enzyme intermediate (B), and activation of conserved water by Glu166 for product release (C).

Scheme 4

BlaC acylation of nitrocefin, cefoxitin, and meropenem. Pyrroline ring isomerization of meropenem results in the incorporation of a solvent exchangeable proton (red) at C3.