Elucidation of mechanism of inhibition and x-ray structure of the TEM-1 beta-lactamase from Escherichia coli inhibited by a N-sulfonyloxy-beta-lactam

Class A beta-lactamases are inactivated by a novel type of monocyclic beta- lactams described recently (J. Am. Chem. Sec. 1995, 117, 5938). A compound of this class, (+/-)-trans-1-N-(tosyloxy)-3-(1-hydroxyethyl)-4-phenyl-2-aze tidinone, is synthesized, is shown to acylate the active site of the TEM-1 beta-lactamase from Escherichia coli rapidly, and resists deacylation for s everal days. The crystal structure of the enzyme-inhibitor complex was dete rmined at 1.95 Angstrom resolution. The features of the three-dimensional s tructure of this acyl-enzyme species and mechanistic studies revealed that a fragmentation of the inactivator ensued on acylation of the active-site s erine and that the ester carbonyl oxygen was outside the oxyanion hole. Thi s ester carbonyl makes a strong hydrogen bond to the protonated form of the side chain of Glu-166, the general base for deacylation of the typical acy l-enzyme intermediates in the normal catalytic process. Furthermore, intera ctions within the active site mandated the existence of the former beta-lac tam amine as an imine or a ketone, and not as an enamine or an enol, and sh ed light on the unique mechanism of action of these enzyme inactivators. Th is type of inactivator holds the promise of application for inhibition of o ther enzymes.