InhA, the NADH-dependent enoyl-ACP reductase from Mycobacterium tuberculosis, is the target for the antitubercular drug isoniazid. The enzyme is involved in the biosynthesis of fatty acids and enzyme inhibition interferes with the synthesis of mycolic acid, a critical component of the mycobacterial cell wall. Resistance to existing antitubercular drugs is a major problem in the spread of tuberculosis and currently 20 percent of M. tuberculosis strains are resistant to isoniazid. Isoniazid resistance results from mutations in the InhA protein or mutations in the metabolic pathway for isoniazid activation and, consequently, InhA is a bona fida target for the development of novel antitubercular agents. The objective of this proposal is to provide detailed information on the mechanism of InhA with the knowledge that this information will be critical to the design and development of drugs that are effective against both sensitive and drug-resistant strains of M. tuberculosis. Initial efforts are focused on using conjugated substrate analogs to probe the mechanism of InhA. Raman and infrared spectroscopy will provide precise information concerning the geometry and electronic structure of substrates, substrate analogs and cofactors bound to InhA. The vibrational studies will elucidate whether the enzyme promotes catalysis by activating the ground state of the substrate or cofactor to hydride transfer. Site-directed mutagenesis, in concert with the spectroscopic studies, will be used to identify and quantitate the role of specific animo acid residues in substrate activation and transition state stabilization. Structural information on the enzyme-bound ligands will also be provided by NMR spectroscopy using transferred NOE methods. The structural data on the bound molecules will be coupled with existing X-ray crystallographic data on the InhA protein to develop a detailed picture of how substrates and inhibitors are bound to InhA. Finally, recent insight into the mechanism of the antibacterial diazaborine inhibitors that target EnvM, the InhA homolog in E. coli, suggests a novel mechanism for the inactivation of InhA by isoniazid. The molecular basis for isoniazid action will be investigated using Raman and NMR spectroscopy.
