Rapid Kinetic and Other Studies of Biological Oxygenases

描述（由申请人提供）：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表征这些反应中的中间体。我们将采用停流动力学和快速冷冻淬灭方法，再加上光谱学，包括紫外可见，EPR，ENDOR和穆斯堡尔方法，以表征反应中发生的中间体。双混合方法，即形成瞬时中间体，然后在规定的时间内与底物或其他化学试剂反应，将用于检查观察到的中间体的反应性。我们最近的研究已经阐明了使我们能够最大限度地形成细胞色素P450 cam的三种高价中间体（化合物I、化合物II样物质和化合物ES）的条件。我们现在计划用上述方法调查这些物种。我们相信，类似的方法也将是有用的邻苯二甲酸酯双加氧酶，催化许多芳香族化合物的微生物好氧代谢的第一步的两个最佳特征的Rieske加氧酶之一的研究。与公共卫生的相关性：加氧酶存在于所有需氧生物体中，并且在类固醇、核酸、儿茶酚胺、胶原、药物、胡萝卜素、木质素和各种外来化合物的生物合成、转化和降解中是重要的。因此，这些酶对大多数有氧生命形式是至关重要的，并且对于处理我们环境中的污染所必需的生物修复过程的发展是必需的，这可能是世界上最严重的长期健康问题。除了它们在生物降解中的作用之外，Rieske非血红素含铁酶的产物通常是顺式-二氢二醇，其在寻求最小化化学污染及其对健康的影响的“绿色”合成化学中有价值。P450酶在药物和其他异生物质物质的代谢中至关重要。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，这些研究的结果将导致更好地理解参与氧合反应的中间体的电子结构，这将是重要的，在了解分子氧是如何被激活的受控代谢过程。这可能反过来导致预测和处理环境中各种化合物代谢的能力提高。

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.