Mechanism of inhibition of the class A beta-lactamases PC1 and TEM-1 by tazobactam - Observation of reaction products by electrospray ionization massspectrometry

The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB ), a potent penicillanic sulfone inhibitor for class A beta-lactamases, wer e studied using electrospray ionization mass spectrometry (ESI/MS). Followi ng inactivation of the beta-lactamases by TZB, new abundant high mass compo nents were observed including three with molecular masses of 52, 70, and 88 Da greater than PC1 and TEM-1, respectively, and a component with a molecu lar mass of 300 Da greater than PC1. In addition, three TZB reaction produc ts with molecular masses of 248, 264, and 280 Da were observed, High perfor mance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct di gested with Glu-C revealed three new components with masses 52, 70, and 88 Da greater than that of the peptide composed of amino acid residues 58-82 a nd one new component with a mass 70 Da greater than that of the peptide com posed of amino acid residues 125-141, HPLC/ESI/MS/MS analysis of the two di gested peptides whose masses increased by 70 Da indicated that Ser-70 and S er-130 were the most likely TZB-modified amino acid residues. Based on thes e data, a mechanism for the inactivation of the class A beta-lactamases by TZB is proposed. In this scheme, initial acylation of Ser-70 by TZB and ope ning of the lactam ring are followed by one of several different events: (1 ) the rapid decomposition of TZB with loss of the enamine moiety to form th e propiolylated enzyme, (2) an intramolecular nucleophilic displacement of the imine or enamine moiety by Ser-130 to form a cross-linked vinyl ether, and (3) hydrolysis of the imine or enamines to form a Ser-70-linked aldehyd e.