Mechanism of inhibition of the PC1 beta-lactamase of Staphylococcus aureus by cephalosporins: importance of the 3'-leaving group

Abstract

The hydrolysis of cephalosporins containing good leaving groups at the 3'-position [those used in this study were the chromogenic cephalosporin PADAC [pyridine-2-azo-4'-(N',N'-dimethylaniline) substituted on cephalosporin], cephaloridine, and cephalothin], catalyzed by the Staphylococcus aureus PC1 beta-lactamase, proceeds in two spectrophotometrically observable phases. The first involves formation of an acyl-enzyme intermediate while the second involves partitioning of this intermediate between two pathways. One path yields the normal cephalosporoate (3) from which the 3'-leaving group is spontaneously eliminated in solution to give the 3-methylenedihydrothiazine 2, while the second involves initial elimination of the 3' substituent, thus yielding a second acyl-enzyme intermediate, which then hydrolyzes to give the same final product as from the first pathway. The second acyl-enzyme is relatively inert to hydrolysis (t1/2 congruent to 10 min at 20 degrees C), and its formation thus leads to transient inhibition of the enzyme. The partition ratio between hydrolysis and elimination at the enzyme active site could be determined either spectrophotometrically from burst experiments or from measurements of residual beta-lactamase activity as a function of cephalosporin concentration. This ratio varied with the leaving group ability of the 3' substituent (acetoxy greater than N,N-dimethylaniline-4-azo-2'-pyridinium greater than pyridinium) in the anticipated fashion. The inert acyl-enzyme intermediate was isolated by exclusion chromatography and shown to contain the cephem nucleus, but not the 3' substituent, covalently bound to the enzyme. As would be expected, PADAC, cephaloridine, and cephalothin yielded the same inert intermediate. Cephalosporins with poor or no 3'-leaving groups, e.g., dansylcephalothin and desacetoxycephalothin, neither displayed the branched pathway nor yielded the long-lived acyl-enzyme.