The mechanism of inactivation of prototypic class A (TEM-1) and class C (Q908R) beta-lactamases by penem BRL 42715 has been investigated by kinetic experiments and modeling computations. Both beta-lactamases are inactivated extremely efficiently with k(inact)/K-i = 10(6)-10(7) M(-1) s(-1) and k(cat)/k(inact) = 1-12. The inhibitor constants were in the nanomolar range. The inactivation chemistry is equally efficient for the Arg-244-Ser mutant of the TEM-1 enzyme, which is resistant to inactivation by the clinical agent clavulanate. The inactivation chemistry is believed to involve acylation of the active-site serine, followed by a rearrangement to a dihydrothiazepine species. Inactivation is reversible in the case of the wild-type and mutant TEM-1 beta-lactamases, but appears irreversible for the Q908R enzyme. Molecular modeling of the initial acylated species and the rearranged dihydrothiazepine species into the active sites of the crystal structures of the two enzymes provided insight into the chemistry of inactivation. Acylation is accompanied by a rotation about the C-5-C-6 bond which is critical for the proper positioning of an incipient thiolate upon its formation from the acyl-enzyme intermediate, en route to the dihydrothiazepine species. Structural information is provided for the reversibility of the inactivated TEM beta-lactamase and the lack of such reversibility for the Q908 enzyme.