Rapid Kinetic and Other Studies of Biological Oxygenases

生物加氧酶的快速动力学和其他研究

• 批准号: 7150067
• 负责人: David P Ballou
• 金额: $ 29.45万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7150067
• 关键词: Mossbauer spectrometry; chemical kinetics; cytochrome P450; electrochemistry; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; electron transport; enzyme mechanism; enzyme substrate; hydrogen bond; iron; metalloenzyme; oxidation reduction reaction; oxygenases; phosphoprotein phosphatase; stop flow technique

DESCRIPTION (provided by applicant): Plan and Goals: The long-term objective of the proposed research is to discover how iron in Rieske non-heme iron oxygenases and in cytochromes P450 oxygenases activates oxygen for reactions with substrates. The specific aims focus on characterizing intermediates in these reactions by rapid kinetics and other physical means. We will employ stopped-flow kinetics and rapid freeze-quench methods, coupled with spectroscopy, including UV-visible, EPR, ENDOR, and Mossbauer methods, to characterize intermediates occurring in the reaction. Double-mixing methods, whereby a transient intermediate is formed and then reacted with a substrate or other chemical agent at a defined time, will be used to examine the reactivity of intermediates observed. Our recent studies have elucidated conditions that will enable us to maximize the formation of three high-valent intermediates with cytochrome P450cam (compound I, compound ll-like species, and compound ES). We now plan to investigate these species by the methods mentioned above. We believe that similar approaches will likewise be useful for studying phthalate dioxygenase, one of the two best-characterized Rieske oxygenases that catalyze the first step in the microbial aerobic metabolism of many aromatic compounds. Relevance to Public Health: Oxygenases are found in all aerobic organisms and are important in the biosynthesis, transformation, and degradation of steroids, nucleic acids, catecholamines, collagen, drugs, prostaglandins, lignin, and various foreign compounds. Thus, these enzymes are crucial to a majority of aerobic life forms and are requisite to the development of bioremediation processes necessary for dealing with pollution in our environment, perhaps the most serious long-term health problem of the world. In addition to their role in biodegradation, the products of Rieske nonheme iron-containing enzymes are often c/s-dihydrodiols, which are valuable in "green" synthetic chemistry that seeks to minimize chemical pollution and its effects on health. P450 enzymes are critical in the metabolism of drugs and other xenobiotic substances. Thus, an understanding of their function is necessary for developing effective pharmaceutical products. We believe that results from these studies will lead to a better comprehension of the electronic structure of intermediates involved in oxygenation reactions, and this will be important in understanding how molecular oxygen is activated for controlled metabolic processes. This may, in turn, lead to an improved ability to predict and deal with the metabolism of various compounds in the environment.

描述(申请人提供)：计划和目标：拟议研究的长期目标是发现Rieske非血红素铁加氧酶和细胞色素P450加氧酶中的铁如何激活氧气与底物反应。具体目标集中在通过快速动力学和其他物理手段来表征这些反应中的中间体。我们将使用停流动力学和快速冷冻急冷法，结合光谱，包括紫外可见、EPR、Endor和穆斯堡尔方法，来表征反应中出现的中间体。双重混合法，即形成一种瞬时中间体，然后在规定的时间与底物或其他化学试剂反应，将用来检查观察到的中间体的反应性。我们最近的研究阐明了使我们能够最大限度地与细胞色素P450cam形成三个高价中间体(化合物I、类化合物11和化合物ES)的条件。我们现在计划用上面提到的方法调查这些物种。我们认为，类似的方法将同样有助于研究邻苯二甲酸酯双加氧酶，这是两种特性最好的Rieske加氧酶之一，催化许多芳香族化合物微生物有氧代谢的第一步。与公共健康相关：加氧酶存在于所有需氧生物体中，对类固醇、核酸、儿茶酚胺、胶原蛋白、药物、前列腺素、木质素和各种外来化合物的生物合成、转化和降解具有重要作用。因此，这些酶对大多数好氧生命形式至关重要，也是开发生物修复过程所必需的，这些过程是处理我们环境中的污染--可能是世界上最严重的长期健康问题--所必需的。除了它们在生物降解中的作用外，Rieske非血红素含铁酶的产品通常是c/S-二氢二醇，它们在寻求将化学污染及其对健康的影响降至最低的“绿色”合成化学中很有价值。P450酶在药物和其他外来物质的新陈代谢中起关键作用。因此，了解它们的功能对于开发有效的药物产品是必要的。我们相信，这些研究的结果将有助于更好地理解参与氧化反应的中间体的电子结构，这对于理解分子氧是如何被激活以进行受控的代谢过程将是重要的。这反过来可能会提高预测和处理环境中各种化合物新陈代谢的能力。