Exploring the Inhibition of CTX-M-9 by β-Lactamase Inhibitors and Carbapenems

Currently, CTX-M beta-lactamases are among the most prevalent and most heterogeneous extended-spectrum beta-lactamases (ESBLs). In general, CTX-M enzymes are susceptible to inhibition by beta-lactamase inhibitors. However, it is unknown if the pathway to inhibition by beta-lactamase inhibitors for CTX-M ESBLs is similar to TEM and SHV beta-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 beta-lactamase was inactivated by sulbactam, tazobactam, clavulanate, meropenem, doripenem, ertapenem, and a 6-methylidene penem, penem 1. K-i values ranged from 1.6 +/- 0.3 mu M(mean +/- standard error) for tazobactam to 0.02 +/- 0.01 mu M for penem 1. Before and after tryptic digestion of the CTX-M-9 beta-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 spectrometry (MALDI-TOF MS) identified different adducts attached to the peptide containing the active site Ser70 (+52, 70, 88, and 156 +/- 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 beta-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 beta-lactamases.