Catalysis of isoniazid action by M tuberculosis KatG

描述(申请人提供)：拟议研究的广泛目标是阐明所提议的途径中的化学步骤，确定过氧化氢酶-过氧化物酶(KatG)在抗结核抗生素异烟肼(INH)作用机制中的作用。这种前药用于治疗结核病已有50多年的历史，但对其作用机制的完整了解仍不清楚。世界各地对异烟肼耐药结核病感染的不断出现要求继续保持警惕，了解抗生素的功能和广泛存在的抗生素耐药性的起源。对结核分枝杆菌KatG结构和催化功能的研究是拟议研究的核心，因为该酶负责药物的“激活”和另一种关键酶的独特抑制剂的产生，该酶是致病分枝杆菌合成霉菌酸和细胞壁完整性所必需的一种烯醇还原酶。该抑制剂是一种由KatG催化的氧化反应生成的酰基-NADH加合物。导致加合物产生的反应的机理和动力学是拟议研究的目标之一。关于基于血红素的催化和酪氨酸基在KatG中的催化的潜在作用的假说将被测试。异烟肼和腺嘌呤二核苷酸作为底物的参与将使用光学停止流动分光光度法和快速冷冻猝灭电子顺磁共振(EPR)进行研究。野生型酶的结果将与几种已知具有1NH耐药性的突变酶的结果进行比较，以确定KatG酶学中耐药性的来源。在拟议的研究中用于研究酶结构和催化机理的其他工具包括共振拉曼光谱和X射线结晶学。等温滴定量热法也被应用于研究KatG酶和INH分子中对药物高亲和力结合的要求。这项研究将为了解一种知之甚少但至关重要的抗结核病药物提供新的见解。

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.