ROLE OF THE OMEGA-LOOP IN THE ACTIVITY, SUBSTRATE-SPECIFICITY, AND STRUCTURE OF CLASS-A BETA-LACTAMASE

The structure of class A beta-lactamases contains an Omega-loop associ ated with the active site, which carries a key catalytic residue, Glu1 66. A 16-residue Omega-loop deletion mutant of beta-lactamase from Sta phylococcus aureus PCl, encompassing residues 163-178, was produced in order to examine the functional and structural role of the loop. The crystal structure was determined and refined at 2.3 Angstrom, and the kinetics of the mutant enzyme was characterized with a variety of beta -lactam antibiotics. In general, the wild-type beta-lactamase hydrolyz es penicillin compounds better than cephalosporins. In contrast, the d eletion of the Omega-loop led to a variant enzyme that acts only on ce phalosporins, including third generation compounds. Kinetic measuremen ts and electrospray mass spectrometry revealed that the first and thir d generation cephalosporins form stable acyl-enzyme complexes, except for the chromogenic cephalosporin, nitrocefin, which after acylating t he enzyme undergoes hydrolysis at a 1000-fold slower rate than that wi th wild-type beta-lactamase. Hydrolysis of the acyl-enzyme adducts is prevented because the deletion of the Omega-loop eliminates the deacyl ation apparatus comprising Glu166 and its associated nucleophilic wate r site. The crystal structure reveals that while the overall fold of t he mutant enzyme is similar to that of the native beta-lactamase, loca l adjustments in the vicinity of the missing loop occurred. The altere d beta-lactam specificity is attributed to these structural changes. I n the native structure, the Omega-loop restricts the conformation of a beta-strand at the edge of the active site depression. Removal of the loop provides the beta-strand with a new degree of conformational fle xibility, such that it is displaced inward toward the active site spac e. Modeled Michaelis complexes with benzylpenicillin and cephaloridine show that the perturbed conformation of the beta-strand is inconsiste nt with penicillin binding because of steric clashes between the beta- lactam side chain substituent and the beta-strand. In contrast, no cla shes occur upon cephalosporin binding. Recognition of third generation cephalosporins is possible because the bulky side chain substituents of the beta-lactam ring typical of these compounds can be accommodated in the space freed by the deletion of the Omega-loop.