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

• Published in: Biochimica et biophysica acta Vol. 1547; no. 2; pp. 196 - 205
• Main Authors: Bonomo, Robert A., Liu, Jingzhou, Chen, Yonghong, Ng, Lily, Hujer, Andrea M., Anderson, Vernon E.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 11.06.2001
• Subjects: Amino Acid Sequence; beta-Lactamase Inhibitors; beta-Lactamases - chemistry; CMY-2 β-lactamase; Electrospray ionization-mass spectroscopy; Enzyme Inhibitors - pharmacology; Mass Spectrometry - methods; Models, Chemical; Molecular Sequence Data; Penicillanic Acid - analogs & derivatives; Penicillanic Acid - pharmacology; Tazobactam; Trypsin; Tazobactam; Electrospray ionization-mass spectroscopy; CMY-2 β-lactamase
• ISSN: 0167-4838; 0006-3002; 1879-2588
• DOI: 10.1016/S0167-4838(01)00175-3

The CMY-2 β-lactamase, a plasmid determined class C cephalosporinase, was shown to be susceptible to inhibition by tazobactam ( K i=40 μM). The reaction product(s) of CMY-2 β-lactamase with the β-lactamase inhibitor tazobactam were analyzed by electrospray ionization/mass spectrometry (ESI/MS) to characterize the prominent intermediates of the inactivation pathway. The ESI/MS determined mass of CMY-2 β-lactamase was 39 851±3 Da. After inactivating CMY-2 β-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 β-lactamase followed by LC/MS identified two 22 amino acid peptides containing the active site Ser64 modified by a fragment of tazobactam. These two peptides were increased in mass by 70 and 88 Da, respectively. UV difference spectra following inactivation revealed the presence of a new species with a 302 nm λ max. Based upon the increase in molecular mass of the tazobactam inactivated CMY-2 β-lactamase, we propose that during the inactivation of this β-lactamase by tazobactam an imine is formed. Tautomerization forms the spectrally observed enamine. Hydrolysis generates the covalently attached malonyl semialdehyde, its hydrate, or an enol. This work provides information on the mass of a stable enzyme intermediate of a class C β-lactamase inactivated by tazobactam and, for the first time, unequivocal evidence that a cross-linked species is not required for apparent inactivation.