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This chapter discusses the mechanism of action of β-lactamases using electrospray ionization mass spectrometry (ESI MS). Initial experiments with the class C β-lactamase from Enterobacter cloacae P99 and the β-lactam antibiotic carbenicillin lead to the direct observation of an acyl-enzyme intermediate 6 and its time course turnover. In a study described in the chapter, wild type β-lactamase 1 was expressed in Bacillus subtilis prophage φ 105MU331. ESI mass spectrometric analysis of both mutants after reaction with showed a mass shift of +199 Da., as was expected for the formation of an enzyme-bound dihydrothiazine derivative. This was confirmed by the UV absorption of the product. Two mechanisms have been proposed for the formation of by wild type β-lactamase 1. Pathway A after the initial opening of the β-lactam ring involves the active site Glu-166 as a nucleophile displacing the 6-β-iodo substituent followed by carboxylate displacement in a trans process by the ring sulfur during the ring expansion step. Pathway β requires rotation about the 5-6 bond after initial opening of the β-lactam ring, followed by trans displacement of the 6-β-iodo substituent by the ring sulfur in the ring expansion step. © 1994, ACADEMIC PRESS, INC.

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Journal article

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39 - 47