The carbapenem family of ��-lactam antibiotics displays a remarkably broad spectrum of bactericidal activity, exemplified by meropenem's phase II clinical trial success in patients with pulmonary tuberculosis, a devastating disease for which ��-lactam drugs historically have been notoriously ineffective. The discovery and validation of l,d-transpeptidases (Ldts) as critical drug targets of bacterial cell-wall biosynthesis, which are only potently inhibited by the carbapenem and penem structural classes, gave an enzymological basis for the effectiveness of the first antitubercular ��-lactams. Decades of study have delineated mechanisms of ��-lactam inhibition of their canonical targets, the penicillin-binding proteins; however, open questions remain regarding the mechanisms of Ldt inhibition that underlie programs in drug design, particularly the optimization of kinetic behavior and potency. We have investigated critical features of mycobacterial Ldt inhibition and demonstrate here that the covalent inhibitor meropenem undergoes both reversible reaction and nonhydrolytic off-loading reactions from the cysteine transpeptidase Ldt through a high-energy thioester adduct. Next-generation carbapenem optimization strategies should minimize adduct loss from unproductive mechanisms of Ldt adducts that reduce effective drug concentration.
