ENZYMATIC CHARACTERIZATION OF THE TARGET FOR ISONIAZID IN MYCOBACTERIUM-TUBERCULOSIS

The inhA gene has been recently shown to encode a common protein targe t for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein ca talyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential fo r fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycoli c acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the p referred ordered addition of NADH and the enoyl substrate. The chemica l mechanism involves stereospecific hydride transfer of the 4S hydroge n of NADH to the C-3 position of the 2-trans-enoyl substrate, followed by protonation at C-2 of an enzyme-stabilized enolate intermediate. K inetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both ison iazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA-N ADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration micr ocalorimetry, suggesting that this compound is a prodrug, which must b e converted to its active form.