A MECHANISTIC STUDY OF SOME O2 OXIDOREDUCTASES

这项研究的长期目标是详细确定各种 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（包括：肌醇 儿茶酚、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），是 认为，更详细地了解其机制， 对许多代谢缺陷的控制或减轻。 三种酶 下面列出的将在未来的赠款期间进行调查，但 重点放在第一个方面。 猪肾肌肌醇加氧酶。 这种酶负责 第一个承诺的步骤肌醇catalysts在动物和已知的是 糖尿病动物体内缺乏。 由于肌醇衍生物现在 已知作为各种激素的第二信使，一项关于 部分负责细胞肌醇状态的酶 似乎是有道理的。 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，并有望获得 关于酶的详细结构信息。 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，获得酶的完整氨基酸序列， 克隆和测序酶的cDNA，表征金属离子， 它的结合位点，希望获得适合完整X射线的晶体 结构测定等。 4-来自猪肝的羟苯丙酮酸双加氧酶。 这种酶， 参与所有动物的酪氨酸代谢， 几种代谢性疾病 大多数拟议的实验都是针对 识别反应中可能的中间体或氨基酸 活性位点的残基。 牛肾D-天冬氨酸氧化酶。 据信这种过氧化物酶体 来控制动物的新陈代谢。 我们的目标是 建议的研究是通过动力学方法（特别是采用 停止流动技术）的解释，一个不寻常的激活，这 单体黄素酶。

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.