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: 7257230
• 负责人: David P Ballou
• 金额: $ 28.59万
• 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/7257230
• 关键词: Address; Aerobic; Affect; Alkanes; Anabolism; Aromatic Compounds; Aromatic Hydrocarbons; Binding; Biodegradation; Biological; Bioremediations; Boston; Camphor; Camphor 5-Monooxygenase; Catalysis; Catecholamines; Cations; Cell Respiration; Characteristics; Chemical Agents; Chemicals; Collaborations; Collagen; Complex; Comprehension; Condition; Coupled; Coupling; Cytochrome P450; Cytochromes; Data; Development; Dioxygenases; Distal; Electron Nuclear Double Resonance; Electron Transport; Electronics; Environment; Enzymes; Freezing; Goals; Hand; Health; Heme Iron; Hydrocarbons; Hydrogen Bonding; Hydrogen Peroxide; Illinois; Iron; Kinetics; Laboratories; Lead; Life; Lignin; Maps; Metabolism; Methods; Michigan; Molecular; Mononuclear; Mossbauer Spectroscopy; Mutation; Nature; Nitrogen; Nucleic Acids; Optics; Organism; Oxidation-Reduction; Oxygen; Oxygenases; Peroxidase; Peroxidases; Peroxides; Personal Satisfaction; Pharmaceutical Preparations; Pharmacologic Substance; Pollution; Porphyrins; Positioning Attribute; Process; Progress Reports; Property; Prostaglandins; Public Health; Publications; Range; Reaction; Reducing Agents; Relative (related person); Research; Research Design; Research Personnel; Roentgen Rays; Role; Scanning; Spectrum Analysis; Steroids; Structure; Sulfur; Synthesis Chemistry; System; Taurine; Temperature; Testing; Thinking; Time; Universities; Variant; Work; Xenobiotics; carboxylate; chemical property; cytochrome P-450 CYP119 (Sulfolobus solfataricus); drug metabolism; experience; improved; microbial; naphthalene dioxygenase; oxidation; phthalate 4,5-dioxygenase; programs

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（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与底物反应的氧气。具体目的集中在表征中间体在这些反应通过快速动力学和其他物理手段。我们将采用停止流动动力学和快速冷冻淬火方法，结合光谱，包括uv -可见，EPR， ENDOR和穆斯堡尔方法，来表征反应中发生的中间体。双重混合方法，即形成一种瞬态中间体，然后在规定的时间内与底物或其他化学试剂发生反应，将用于检查所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三种具有细胞色素P450cam的高价中间体的条件（化合物I，化合物ll-like species和化合物ES）。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有助于邻苯二甲酸酯双加氧酶的研究，邻苯二甲酸酯双加氧酶是两种最具特征的Rieske加氧酶之一，它催化了许多芳香族化合物的微生物有氧代谢的第一步。与公共卫生相关：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、转化和降解中起重要作用。因此，这些酶对大多数有氧生命形式是至关重要的，也是发展处理环境污染所必需的生物修复过程所必需的，环境污染可能是世界上最严重的长期健康问题。除了在生物降解中发挥作用外，Rieske非血红素含铁酶的产物通常是c/s-二氢二醇，这在寻求尽量减少化学污染及其对健康影响的“绿色”合成化学中很有价值。P450酶在药物和其他外源物质的代谢中起关键作用。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氧化反应中中间体的电子结构，这对于理解分子氧如何被激活以控制代谢过程将是重要的。反过来，这可能会提高预测和处理环境中各种化合物代谢的能力。