PROBING THE NON-PROLINE CIS PEPTIDE-BOND IN BETA-LACTAMASE FROM STAPHYLOCOCCUS-AUREUS PC1 BY THE REPLACEMENT ASN136-]ALA

A non-proline cis peptide is present between Glu166 and Ile167 in the active site of beta-lactamase from Staphylococcus nul eus PC1. To exam ine the role of the interaction between the side chain of Asn136 and t he main chain of Glu166, the site-directed mutant N136A was produced. The enzyme shows no measurable hydrolytic activity toward a variety of penicillins or cephalosporins except for the chromogenic cephalospori n, nitrocefin. For nitrocefin, the progress curve exhibits a fast burs t with a stoichiometry of 1 mol of degraded substrate per mole of enzy me followed by a slow phase with a hydrolysis rate that is reduced by approximately 700-fold compared with that of the wild-type enzyme. Thu s, the mutant enzyme is deacylation defective. Monitoring the hydrolys is of nitrocefin after preincubation with a number of beta-lactam comp ounds shows that cephalosporins form stable acyl complexes with the en zyme, whereas penicillins do not. The molecular weight of the mutant w as determined by electrospray mass spectrometry, and the presence of t he stable acyl enzyme adducts with cephaloridine and cefotaxime was co nfirmed by both electrospray and MALDI mass spectrometry. Therefore, i n addition to impairing deacylation, the acylation machinery has been altered compared with the wild-type enzyme to act on cephalosporins an d not on penicillins. Urea denaturation and thermal unfolding studies show that the N136A mutant enzyme is less stable than the wild-type en zyme. However, stability against chemical denaturation of the mutant e nzyme is enhanced in the presence of cephaloridine beyond the stabilit y of the wild-type protein. This is attributed to accumulation of favo rable interactions between the cephaloridine and the protein, which pl ay a role in the folded state and not in the unfolded state.