Inactivation of CMY-2 beta-lactamase by tazobactam: initial mass spectroscopic characterization

The CMY-2 p-lactamase, a plasmid determined class C cephalosporinase, was s hown to be susceptible to inhibition by tazobactam (K-i = 40 muM). The reac tion product(s) of CMY-2 beta -lactamase with the beta -lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI /MS) to characterize the prominent intermediates of the inactivation pathwa y. The ESI/MS determined mass of CMY-2 beta -lactamase was 39 851 +/- 3 Da. After inactivating CMY-2 beta -lactamase with excess tazobactam, a single species, M-r= 39 931 +/- 3.0, was detected. Comparison of the peptide maps from tryptic digestion of the native enzyme and the inactivated p-lactamase followed by LC/MS identified two 22 amino acid peptides containing the act ive site Ser64 modified by a fragment of tazobactam. These two peptides wer e increased in mass by 70 and 88 Da, respectively. UV difference spectra fo llowing inactivation revealed the presence of a new species with a 302 nm l ambda (max). Based upon the increase in molecular mass of the tazobactam in activated CMY-3 beta -Lactamase, we propose that during the inactivation of this beta -lactamase by tazobactam an imine is formed. Tautomerization for ms the spectrally observed enamine. Hydrolysis generates the covalently att ached malonyl semialdehyde, its hydrate, or an enol. This work provides inf ormation on the mass of a stable enzyme intermediate of a class C beta -lac tamase inactivated by tazobactam and, for the first time, unequivocal evide nce that a cross-linked species is not required for apparent inactivation. (C) 2001 Elsevier Science B.V. All rights reserved.