THE CATALYTIC MECHANISM OF BETA-LACTAMASES - NMR TITRATION OF AN ACTIVE-SITE LYSINE RESIDUE OF THE TEM-1 ENZYME

beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detai led structural and mechanistic understanding of these enzymes can be e xpected to guide the design of new antibacterial compounds resistant t o their action. A number of high-resolution structures are available f or class A beta-lactamases, whose catalytic mechanism involves the acy lation of a serine residue at the active site. The identity of the gen eral base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine re sidue and a glutamic acid residue having been proposed as candidates f or this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene H-1 and C-13 chemical shifts (in e nzyme selectively labeled with [epsilon-C-13]lysine) to estimate the p K(a) of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamas e. Both methods show that the pK(a) of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in cat alysis. An alternative mechanism in which this role is performed by gl utamate-166 through an intervening water molecule is described.