CRYSTAL-STRUCTURE OF AN ACYLATION TRANSITION-STATE ANALOG OF THE TEM-1 BETA-LACTAMASE - MECHANISTIC IMPLICATIONS FOR CLASS-A BETA-LACTAMASES

The crystal structure of a phosphonate complex of the class A TEM-1 be ta-lactamase has been determined to a resolution of 2.0 Angstrom. The phosphonate appears stoichiometrically at the active site, bound coval ently to Ser70O gamma, with one phosphonyl oxygen in the oxyanion hole . Although the overall structure is very similar to that of the native enzyme (rms difference 0.37 Angstrom for all heavy atoms), changes ha ve occurred in the position of active site functional groups, The acti ve site is also not in the conformation observed in the complex of ano ther class A beta-lactamase, that of Staphylococcus aureus PCl, with t he same phosphonate [Chen, C. C. H., et al, (1993) J. Mel. Biol, 234,1 65-178]. Both phosphonate structures, however, can be seen to represen t models of acylation transition-states since in each the deacylating water molecule appears firmly bound to the Glu166 carboxylate group, T he major difference between the structures lies in the positioning of Lys73N xi and Ser130O gamma. In the S. aureus structure, the closest i nteraction of these functional groups is between Lys73N xi and Ser70O gamma (2.8 Angstrom), while in the TEM-I structure it is between Ser13 0O gamma and the second phosphonyl oxygen of the bound inhibitor (2.8 Angstrom). The former structure therefore may resemble a transition st ate for formation of the tetrahedral species in acylation by nucleophi lic attack on the substrate, where Lys73N xi presumably catalyzes the reaction as a general base. The TEM-1 structure can then be seen as an analogue of the transition state for breakdown of the tetrahedral spe cies, where Ser130O gamma is acting as a general acid, assisting the d eparture of the leaving group. The class A beta-lactamase crystal stru ctures now available lead to a self-consistent proposal for a mechanis m of catalysis by these enzymes.