Catalysis of isoniazid action by M tuberculosis KatG

描述（由申请人提供）：拟议研究的主要目标是阐明确定过氧化氢酶-过氧化物酶（KatG）在抗结核抗生素异烟肼（INH）作用机制中的作用的拟议途径中的化学步骤。这种前药用于治疗结核病已有50多年的历史，但其作用机制的全貌仍不清楚。世界各地不断出现耐inh结核感染，需要继续保持警惕，了解抗生素的功能和广泛的抗生素耐药性的起源。研究结核分枝杆菌KatG的结构和催化功能是这项研究的核心，因为这种酶负责“激活”药物和产生另一种关键酶的独特抑制剂，这种酶是致病性分枝杆菌中霉菌酸生物合成和细胞壁完整性所必需的一种烯基还原酶。该抑制剂是由KatG催化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的之一。关于血红素基催化和酪氨酸自由基催化在KatG中的潜在作用的假设将被测试。INH和腺嘌呤二核苷酸作为底物的参与将使用光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已知具有1NH抗性的几种突变酶的结果进行比较，以确定KatG酶学中耐药的起源。其他用于研究酶结构和催化机理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。这项研究将为一种知之甚少但极其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.