A MECHANISTIC STUDY OF SOME O2 OXIDOREDUCTASES

一些O2氧化还原酶的机理研究

• 批准号: 3225052
• 负责人: GORDON A HAMILTON
• 金额: $ 11.86万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-05-01 至 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3225052
• 关键词: X ray crystallography; aminoacid oxidase; analytical method; complementary DNA; cow; electron spin resonance spectroscopy; enzyme complex; enzyme mechanism; enzyme structure; flavoproteins; high performance liquid chromatography; inositol; kidney; liver; molecular cloning; oxidoreductase; oxygenases; radiotracer; swine; tyrosine

The long term goal of this research is to determine in detail how various O2 oxidoreductases catalyze reactions of O2. Since these enzymes are necessary for so many important metabolic pathways (including: inositol catabolism, aromatic amino acid metabolism, catecholamine biosynthesis and degradation, steroid hormone biosynthesis, drug metabolism, etc.), it is felt that a more detailed understanding of their mechanisms could lead to the control or alleviation of many metabolic defects. The three enzymes listed below will be investigated during the coming grant period, but the major emphasis will be on the first. myo-Inositol oxygenase from pig kidney. This enzyme is responsible for the first committed step in inositol catabolism in animals and is known to be deficient in animals with diabetes. Since inositol derivatives are now known to act as second messengers for various hormones, a detailed study of the enzyme that is partially responsible for the inositol status of cells seems warranted. A major focus of the proposed work will be to identify and characterize enzymic groups involved in catalysis, and hopefully obtain detailed structural information about the enzyme. To this end we plan to: further characterize and develop affinity labels for the enzyme's active site residues, obtain the complete amino acid sequence of the enzyme by cloning and sequencing the enzyme's cDNA, characterize the metal ion and its binding site, hopefully obtain crystals suitable for a complete X-ray structure determination, etc. 4-Hydroxyphenylpyruvate dioxygenase from pig liver. This enzyme, which participates in the metabolism of tyrosine in all animals, is involved in several metabolic diseases. Most of the proposed experiments are aimed at identifying, either possible intermediates in the reaction, or amino acid residues at the active site. D-Aspartate oxidase from beef kidney. This peroxisomal enzyme is believed to function in the control of metabolism in animals. Our goal in the proposed research is to determine by kinetic methods (especially employing stopped flow techniques) the explanation for an unusual activation of this monomeric flavoenzyme by some effectors.

这项研究的长期目标是详细确定不同的 O2 氧化还原酶催化 O2 的反应。 由于这些酶是 许多重要的代谢途径所必需的（包括：肌醇 分解代谢、芳香族氨基酸代谢、儿茶酚胺生物合成和 降解、类固醇激素生物合成、药物代谢等）， 认为更详细地了解其机制可能会导致 控制或减轻许多代谢缺陷。 三种酶 下面列出的将在即将到来的资助期内进行调查，但是 主要重点是第一个。 来自猪肾的肌醇加氧酶。 这种酶负责 动物体内肌醇分解代谢的第一个关键步骤是 患有糖尿病的动物缺乏。 由于肌醇衍生物现在 已知可作为各种激素的第二信使，一项详细研究 部分负责细胞肌醇状态的酶 似乎有道理。 拟议工作的一个主要重点是确定 并表征参与催化的酶基团，并希望获得 有关酶的详细结构信息。 为此，我们计划： 进一步表征和开发酶活性的亲和标记 位点残基，通过以下方式获得酶的完整氨基酸序列 克隆并测序酶的 cDNA，表征金属离子并 其结合位点，有望获得适合完整X射线的晶体 结构测定等 来自猪肝的 4-羟基苯丙酮酸双加氧酶。 这种酶， 参与所有动物的酪氨酸代谢，参与 几种代谢性疾病。 大多数提议的实验旨在 识别反应中可能的中间体或氨基酸 活性位点上的残留物。 来自牛肾的 D-天冬氨酸氧化酶。 这种过氧化物酶体酶被认为 发挥控制动物新陈代谢的作用。 我们的目标是 拟议的研究是通过动力学方法（特别是采用 停流技术）对此异常激活的解释 单体黄素酶通过一些效应器。