Action Mechanism of Antitubercular Isoniazid

Abstract

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an in- hibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the in- hibitor 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 A326/A278 ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a Kd < 0.4 nM and can be dissociated from denatured InhA for chromato- graphic isolation. The free inhibitor showed absorption peaks at 326 ( 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, Mn2-mediated re- action based on high pressure liquid chromatography analysis and absorption and mass spectral characteris- tics. 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. Further- more, the formation of the InhA inhibitor in the KatG reaction was independent of InhA.