Action mechanism of antitubercular isoniazid. Activation by Mycobacterium tuberculosis KatG, isolation, and characterization of inha inhibitor

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristic A(326)/A(278) ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K(d) < 0.4 nM and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (epsilon(326) 6900 M(-1) cm(-1)) and 260 nm (epsilon(260) 27,000 M(-1) cm(-1)). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn(2+)-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.