Exploring the Inhibition of CTX-M-9 by (cid:1) -Lactamase Inhibitors and Carbapenems (cid:1) †

Currently, CTX-M (cid:1) -lactamases are among the most prevalent and most heterogeneous extended-spectrum (cid:1) -lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by (cid:1) -lactamase inhibitors. However, it is unknown if the pathway to inhibition by (cid:1) -lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV (cid:1) -lactamases and why bacteria possessing only CTX-M ESBLs are so susceptible to carbapenems. Here, we have performed a kinetic analysis and timed electrospray ionization mass spectrometry (ESI-MS) studies to reveal the intermediates of inhibition of CTX-M-9, an ESBL representative of this family of enzymes. CTX-M-9 (cid:1) -lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K i values ranged from 1.6 (cid:2) 0.3 (cid:3) M (mean (cid:2) standard error) for tazobactam to 0.02 (cid:2) 0.01 (cid:3) M for penem 1. Before and after tryptic digestion of the CTX-M-9 (cid:1) -lactamase apo-enzyme and CTX-M-9 inactivation by inhibitors (meropenem, clavulanate, sulbactam, tazobactam, and penem 1), ESI-MS and matrix-assisted laser desorption ionization–time of flight mass spectrom- etry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 ( (cid:4) 52, 70, 88, and 156 (cid:2) 3 atomic mass units). This study shows that a multistep inhibition pathway results from modification or fragmentation with clavulanate, sulbactam, and tazobactam, while a single acyl enzyme intermediate is detected when meropenem and penem 1 inactivate CTX-M-9 (cid:1) -lactamase. More generally, we propose that Arg276 in CTX-M-9 plays an essential role in the recognition of the C 3 carboxylate of inhibitors and that the localization of this positive charge to a “region of the active site” rather than a specific residue represents an important evolutionary strategy used by (cid:1) -lactamases. extended-spectrum The kinetic parameters of hydrolysis and inhibition of CTX-M-9 (cid:1) -lactamase were determined by continuous assays at room temperature (RT) using a diode array spectrophotometer (Agilent model 8453) in the manner described previ- ously (22, 53). Each assay was performed in a 1-ml quartz cuvette with 10 mM phosphate-buffered saline at pH 7.4. Measurements were obtained using NCF ( (cid:4) 482 of 17,400 M (cid:5) 1 cm (cid:5) ). Enzfitter (Biosoft Cor-poration) energy minimization of each ligand pose was performed using the program CDOCKER (61). The molecule was immersed in a water box, 7 Å from any face of the box, using the Solvation module of DS 2.1 with explicit periodic boundary conditions (PBC). All models were further minimized and equilibrated using a standard dynamic cascade protocol (minimization with steepest descent and minimization with conjugate gradient followed by a 6-ps dynamic simulation with heating, equilibration, and production steps). All energy minimizations and molecular dynamics simulations (MDS) of the enzyme and enzyme complexes were carried out using force field parameters of CHARMm. The Particle Mesh Ewald (PME) method was used to take into account long-range electrostatics, and the bonds that involved hydrogen atoms were constrained with the SHAKE algorithm.