The broad objectives of the proposed research are to elucidate the chemical steps in a proposed pathway defining the role of catalase-peroxidase (KatG) in the mechanism of action of isoniazid (INH), an anti-tuberculosis antibiotic. This pro-drug has been in use for over 50 years to treat TB yet a complete picture of its mechanism of action is still not clearly understood. The continuing emergence of INH-resistant TB infection throughout the world demands continued vigilance in learning about antibiotic function and the origins of widespread antibiotic resistance. The investigation of the structure and catalytic function of M. tuberculosis KatG is central to the proposed research as this enzyme is responsible for """"""""activation"""""""" of the drug and production of a unique inhibitor of another key enzyme, an enoyl reductase required for mycolic acid biosynthesis and cell wall integrity in pathogenic mycobacteria. This inhibitor is an acyl-NADH adduct generated through oxidative reactions catalyzed by KatG. The mechanism and kinetics of the reactions leading to production of the adduct are among the aims in the proposed research. Hypotheses about the potential role of heme-based catalysis and catalysis by a tyrosyl radical in KatG will be tested. The participation of both INH and adenine dinucleotides as substrates will be investigated using optical-stopped flow spectrophotometry and rapid freeze-quench electron paramagnetic resonance (EPR). Results for the wild-type enzyme will be compared to those for several mutant enzymes known to confer 1NH resistance to identify the origins of drug resistance in KatG enzymology. Other tools used in the proposed research to study enzyme structure and catalytic mechanism include resonance Raman spectroscopy and x-ray crystallography. The technique of isothermal titration calorimetry is also being applied to study the requirements in the KatG enzyme and in the INH molecule for high affinity binding of the drug. The research will provide new insights into a poorly understood but critically important anti-TB agent.
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