EVIDENCE FOR A NEW ENZYME-CATALYZED REACTION OTHER THAN BETA-LACTAM HYDROLYSIS IN TURNOVER OF A PENEM BY THE TEM-1 BETA-LACTAMASE

The mechanism of turnover of the penem Sch 34343 by the TEM-1 beta-lac tamase, a class A beta-lactamase, has been investigated. In contrast w ith carbapenems, Sch 34343 does not inhibit the beta-lactamase in the course of turnover and the kinetics were not biphasic. Sch 34343 is co nverted to a compound A, which subsequently gives rise to a compound B ; both conversions were mediated by the beta-lactamase. The two compou nds were purified, and their structures were determined. Compound A, t he immediate product of turnover, was shown to share all structural el ements of Sch 34343, except the beta-lactam moiety was hydrolyzed. The structural assignment of this compound constitutes the first direct r eport for the existence of ''penemoic acid' as a product of hydrolysis of penems by beta-lactamases. Compound B was shown to be -mercapto-3- [[2-(carbamoyloxy)ethyl]thio]propenoic acid (4). The conversion of com pound A to compound B by beta-lactamase was active site-directed, the rates showed saturation, and a rate enhancement of 10(7)-fold was meas ured for this reaction compared to the corresponding nonenzymic buffer -catalyzed reaction. Two plausible mechanisms for this unprecedented r eaction for beta-lactamases are offered.