Mechanism of inhibition of RTEM-2 beta-lactamase by cephamycins: relative importance of the 7 alpha-methoxy group and the 3' leaving group.

Cefoxitin is a poor substrate of many beta-lactamases, including the RTEM-2 enzyme. Fisher and co-workers [Fisher, J., Belasco, J. G., Khosla, S., & Knowles, J. R. (1980) Biochemistry 19, 2895-2901] showed that the reaction between cefoxitin and RTEM-2 beta-lactamase yielded a moderately stable acyl-enzyme whose hydrolysis was rate-determining to turnover at saturation. The present work shows first that the covalently bound substrate in this acyl-enzyme has a 5-exo-methylene-1,3-thiazine structure, i.e., that the good (carbamoyloxy) 3' leaving group of cefoxitin has been eliminated in formation of the acyl-enzyme. Such an elimination has recently been shown in another case to yield an acyl-beta-lactamase inert to hydrolysis [Faraci, W. S., & Pratt, R. F. (1985) Biochemistry 24, 903-910]. Thus the cefoxitin molecule has two potential sources of beta-lactamase resistance, the 7 alpha-methoxy group and the good 3' leaving group. That the latter is important in the present example is shown by the fact that with analogous substrates where no elimination occurs at the enzyme active site, such as 3'-de(carbamoyloxy)cefoxitin and 3'-decarbamoylcefoxitin, no inert acyl-enzyme accumulates. An analysis of the relevant rate constants shows that the 7 alpha-methoxy group weakens noncovalent binding and slows down both acylation and deacylation rates, but with major effect in the acylation rate, while elimination of the 3' leaving group affects deacylation only.(ABSTRACT TRUNCATED AT 250 WORDS)