Enzymatic Characterization of the Target for Isoniazid in Mycobacterium tuberculosis

Annaik Quémard, James C. Sacchettini, Andréa Dessen, Catherine Vilcheze, Robert Bittman, William R. Jacobs, John S. Blanchard

Research output: Contribution to journalArticlepeer-review

345 Scopus citations

Abstract

The inhA gene has been recently shown to encode a common protein target for isoniazid and ethionamide action in Mycobacterium tuberculosis. In this paper, we demonstrate that the M. tuberculosis InhA protein catalyzes the NADH-specific reduction of 2-trans-enoyl-ACP, essential for fatty acid elongation. This enzyme preferentially reduces long-chain substrates (12-24 carbons), consistent with its involvement in mycolic acid biosynthesis. Steady-state kinetic studies showed that the two substrates bind to InhA via a sequential kinetic mechanism, with the preferred ordered addition of NADH and the enoyl substrate. The chemical mechanism involves stereospecific hydride transfer of the 4S hydrogen of NADH to the C3 position of the 2-trans-enoyl substrate, followed by protonation at C2 of an enzymestabilized enolate intermediate. Kinetic and microcalorimetric analysis demonstrates that the binding of NADH to the S94A mutant InhA, known to confer resistance to both isoniazid and ethionamide, is altered. This difference can account for the isoniazid-resistance phenotype, with the formation of a binary InhA- NADH complex required for drug binding. Isoniazid binding to either the wild-type or S94A mutant InhA could not be detected by titration microcalorimetry, suggesting that this compound is a prodrug, which must be converted to its active form.

Original languageEnglish (US)
Pages (from-to)8235-8241
Number of pages7
JournalBiochemistry
Volume34
Issue number26
DOIs
StatePublished - Jul 1995

ASJC Scopus subject areas

  • Biochemistry

Fingerprint

Dive into the research topics of 'Enzymatic Characterization of the Target for Isoniazid in Mycobacterium tuberculosis'. Together they form a unique fingerprint.

Cite this