Rapid Kinetic and Other Studies of Biological Oxygenases

描述(申请人提供)：这个项目是对加氧酶和其他氧化还原酶的结构和功能的多学科研究。主要的焦点是Rieske加氧酶和细胞色素P450如何激活氧。此外，对其他金属蛋白的合作研究也在进行中。加氧酶存在于所有需氧生物中，在类固醇、核酸、儿茶酚胺、胶原、药物、前列腺素、木质素和各种外来化合物的生物合成、转化和降解中起重要作用。因此，这些酶对大多数好氧生命形式是至关重要的，也是开发处理环境污染所必需的生物修复过程所必需的，环境污染是世界上主要的健康问题之一。我们将详细研究邻苯二甲酸双加氧酶(PDO)的物理、化学和动力学性质，PDO是Rieske加氧酶的范例，催化许多芳香族化合物有氧代谢的第一步。除了它们在生物降解中的作用外，Rieske非血红素含铁酶催化的产物通常是顺式二氢二醇，这在绿色合成化学中是有价值的。我们将对参与氧化反应的中间体进行表征。此外，我们将(与M.J.Coon和J.Dawson合作)研究细胞色素P450参与氧化过程的中间体的几个方面。我们的假设是，针对底物和产物以及涉及的中间体对这两种类型的系统进行平行研究，将有助于加深对这些氧化过程的理解。建议的研究将使用快速动力学光谱、化学猝灭和其他酶学方法。X射线结晶学和基因技术，包括克隆、表达和突变，也将被用来更好地理解蛋白质如何催化这些有趣的反应。我们的方法将是修饰活性中心残基，然后通过各种物理技术研究催化的各个步骤是如何受到影响的。我们相信，这些研究的结果将有助于更好地理解分子氧是如何被激活以进行受控的代谢过程。这反过来可能会提高预测各种化合物在环境中如何代谢的能力。

DESCRIPTION (provided by applicant): This project is a multi-disciplinary study of the structure and function of oxygenases and other redox enzymes. The principle focus is on how oxygen is activated by Rieske oxygenases and on cytochromes P450. In addition, collaborative studies of other metalloproteins are also being carried out. 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, one of the major health problems of the world.We will investigate in detail the physical, chemical, and kinetic properties of phthalate dioxygenase (PDO), a paradigm for the Rieske oxygenases that catalyze the first step in the aerobic metabolism of many aromatic compounds. In addition to their role in biodegradation, the products of Rieske nonheme iron-containing enzyme catalysis are often cis-dihydrodiols, which are valuable in "green" synthetic chemistry. We will characterize intermediates that are involved in the oxygenation reaction. In addition, we will investigate (in collaboration with M.J. Coon and J. Dawson) several aspects of intermediates involved in oxygenation processes by cytochromes P450. Our hypothesis is that parallel studies of these two types of systems with respect to substrates and products, and intermediates involved, will be complementary to developing a deeper understanding of these oxygenative processes. The proposed studies will employ rapid kinetics spectroscopy, chemical quenching, and other enzymological methods. X-ray crystallography and genetic techniques, including cloning, expression, and mutagenesis, will also be used to develop a better understanding of how the proteins catalyze these interesting reactions. Our approach will be to modify active site residues, and then to study by a variety of physical techniques how various steps in catalysis are affected.We believe that results from these studies will lead to a better understanding of how molecular oxygen is activated for controlled metabolic processes. This may, in turn, lead to an improved ability to predict how various compounds will be metabolized in the environment.