Elucidation of mechanism of inhibition and x-ray structure of the TEM-1 β-lactamase from Escherichia coli inhibited by a N-sulfonyloxy-β-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-azetidinone, is synthesized, is shown to acylate the active site of the TEM-1 beta-lactamase from Escherichia coli rapidly, and resists deacylation for several days. The crystal structure of the enzyme-inhibitor complex was determined at 1.95 Angstrom resolution. The features of the three-dimensional structure of this acyl-enzyme species and mechanistic studies revealed that a fragmentation of the inactivator ensued on acylation of the active-site serine and that the ester carbonyl oxygen was outside the oxyanion hole. This 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 acyl-enzyme intermediates in the normal catalytic process. Furthermore, interactions within the active site mandated the existence of the former beta-lactam amine as an imine or a ketone, and not as an enamine or an enol, and shed light on the unique mechanism of action of these enzyme inactivators. This type of inactivator holds the promise of application for inhibition of other enzymes.