Catalysis of isoniazid action by M tuberculosis KatG

描述（由申请方提供）：拟定研究的广泛目的是阐明拟定途径中的化学步骤，该途径定义了过氧化氢酶-过氧化物酶（KatG）在异烟肼（INH）（一种抗结核抗生素）作用机制中的作用。这种前体药物已用于治疗结核病超过50年，但其作用机制的全貌仍不清楚。世界各地持续出现的INH耐药结核病感染要求人们继续警惕，了解抗生素的功能和广泛的抗生素耐药性的起源。对M.结核病KatG是所提出的研究的中心，因为这种酶负责药物的“活化”和另一种关键酶的独特抑制剂的产生，所述另一种关键酶是致病分枝杆菌中分枝菌酸生物合成和细胞壁完整性所需的烯酰还原酶。该抑制剂是通过KatG催化的氧化反应产生的酰基-NADH加合物。导致产生加合物的反应的机理和动力学是所提出的研究的目标之一。血红素为基础的催化和催化的酪氨酸自由基在KatG的潜在作用的假设将进行测试。将使用光学停流分光光度法和快速冷冻淬灭电子顺磁共振（EPR）研究INH和腺嘌呤二核苷酸作为底物的参与。将野生型酶的结果与已知赋予1 NH抗性的几种突变酶的结果进行比较，以确定KatG酶学中耐药性的起源。其他用于研究酶结构和催化机制的工具包括共振拉曼光谱和X射线晶体学。等温滴定量热法的技术也被应用于研究KatG酶和INH分子对药物高亲和力结合的要求。这项研究将为人们对一种知之甚少但至关重要的抗结核药物提供新的见解。

DESCRIPTION (provided by applicant): 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.