Mechanism of inhibition of the class A beta -lactamases PC1 and TEM-1 by tazobactam. Observation of reaction products by electrospray ionization mass spectrometry.
The reactions of class A beta-lactamases PC1 and TEM-1 with tazobactam (TZB), a potent penicillanic sulfone inhibitor for class A beta-lactamases, were studied using electrospray ionization mass spectrometry (ESI/MS). Following inactivation of the beta-lactamases by TZB, new abundant high mass components 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 molecular mass of 300 Da greater than PC1. In addition, three TZB reaction products with molecular masses of 248, 264, and 280 Da were observed. High performance liquid chromatography (HPLC)/ESI/MS analysis of the TZB-PC1 adduct digested 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 and one new component with a mass 70 Da greater than that of the peptide composed of amino acid residues 125-141. HPLC/ESI/MS/MS analysis of the two digested peptides whose masses increased by 70 Da indicated that Ser-70 and Ser-130 were the most likely TZB-modified amino acid residues. Based on these 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 opening 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 the 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 aldehyde.
Yang
Y
Y
Wyeth-Ayerst Research, Pearl River, New York 10965, USA. yangy@war.wyeth.com
Janota
K
K
Tabei
K
K
Huang
N
N
Siegel
M M
MM
Lin
Y I
YI
Rasmussen
B A
BA
Shlaes
D M
DM
D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D013058:Mass Spectrometry / Q000379:methods; D010397:Penicillanic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D012697:Serine Endopeptidases / Q000378:metabolism; D000078142:Tazobactam; D014357:Trypsin / Q000378:metabolism; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D065093:beta-Lactamase Inhibitors; D010397:Penicillanic Acid; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; C054232:beta-lactamase PC1; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D000078142:Tazobactam