The major role of the propose research is to explain the catalytic function of Mycobacterium Tuberculosis catalase-peroxidase, a heme-enzyme, in the activation of the antimycobacterial antibiotic, isoniazid (isonicotinic acid hydrazide).
The specific aims i nclude identification of the proximal ligand to heme iron in catalase-peroxidase, characterization of the spin state and coordination number of the heme iron in the resting enzyme, identification of hypervalent enzyme intermediates, and kinetic analysis of the reaction of these intermediates with isoniazid. Of special interest is the potential catalytic competence of oxy-ferrous catalase- peroxidase, and the potential for peroxynitrite to activate the enzyme. The role of selected amino acid residues in the catalytic mechanism an in isoniazid binding will be evaluated through examination of the properties of two mutant catalase-peroxidase enzymes identified from clinically isolated, isoniazid resistant M. tuberculosis strains. Inhibition of another M. tuberculosis enzyme, a fatty acyl enoyl reductase (the inhA protein) thought to be a target of drug action, by isoniazid activated catalase-peroxidase, will also be investigated.
Other aims address the Mn(II)-peroxidase activity of catalase-peroxidase considered important because Mn(III) is an efficient single electron oxidant of isoniazid. The techniques of optical stopped-flow spectroscopy, resonance Raman, and electron paramagnetic resonance spectroscopies will be applied in the experimental protocols. The results of the proposed studies will advance a detailed understanding of the action of a first line antibiotic in current use to treat tuberculosis.
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