FLAVOENZYME MECHANISMS--REDOX AND NONREDOX REACTIONS

黄素酶机制——氧化还原和非氧化还原反应

• 批准号: 6033049
• 负责人: MARILYN S JORNS
• 金额: $ 20万
• 依托单位: MCP HAHNEMANN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-03 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6033049
• 关键词: Corynebacterium; DNA repair; Escherichia coli; Saccharomyces cerevisiae; active sites; amine oxidase (flavin); carbon carbon lyase; chromophore; cofactor; covalent bond; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; flash photolysis; flavin adenine dinucleotide; flavins; flavoproteins; high performance liquid chromatography; hybrid enzyme; oxidation reduction reaction; pyrimidine dimers; sarcosine; tetrahydrofolates

Our long term objectives are to elucidate the mechanism of oxidation- reduction reactions catalyzed by flavoenzymes and to evaluate the role of flavin in enzymes where catalysis does not involve a net oxidation- reduction reaction. Corynebacterial sarcosine oxidase, an enzyme useful in clinical diagnosis of kidney function, is a heterotetrameric protein ((alpha beta delta gamma) containing covalent (beta subunit) and noncovalent flavins, a single binding site for sarcosine and two sites for another substrate, tetrahydrofolate. The (beta subunit exhibits sequence homology with various monomeric amine oxidoreductases containing only covalent flavin. To determine whether this new family of flavoenzymes also includes mammalian sarcosine dehydrogenase, we plan to sequence the genes for pig and human sarcosine dehydrogenase. Information on the human gene may be useful in elucidating the molecular basis of sarcosinemia, an autosomal recessive disease characterized by a deficiency of sarcosine dehydrogenase, and high levels of sarcosine in plasma and urine, and possibly associated with neurological and other problems. The functional significance of the complex quaternary structure of corynebacterial sarcosine oxidase will be probed in studies to locate binding sites for various ligands and to determine whether all subunits are essential for sarcosine oxidation. We also seek to elucidate the mechanism of electron transfer from sarcosine to the noncovalent flavin, the kinetics and thermodynamics of interflavin electron transfer, and the biosynthesis and catalytic significance of the covalent flavin linkage. We plan to identify the cysteine residue which forms a reversible complex with the covalent flavin in what appears to represent a novel mechanism of enzyme regulation. The mechanism of human sarcosine dehydrogenase, including its interaction with tetrahydrofolate and the role and biosynthesis of the covalent flavin linkage, will be investigated and compared with the more complex corynebacterial enzyme. Another project involves DNA photolyases from Escherichia coil and Saccharomyces cerevisiae. These enzymes contain pterin and reduced flavin chromophores which enable them to use visible light to repair pyrimidine dimers, the major damage caused by exposure of DNA to ultraviolet light. If left unrepaired, this DNA damage can cause mutations, cancer and cell death. The pterin chromophore binds to a N-terminal domain and acts as an antenna, harvesting light energy which is then transferred to the reduced flavin. The reduced flavin binds to a C-terminal domain and its excited singlet state is the species that directly interacts with substrate and initiates DNA repair. We seek to determine the structure of yeast photolyase and to investigate the specificity and importance of interdomain interactions by creating chimeric enzymes. We seek to characterize the flavin environment and any perturbations induced by substrate. We seek to investigate the mechanism of dimer repair in studies involving time-resolved spectroscopic techniques.

我们的长期目标是阐明氧化机制- 还原反应催化的黄素酶，并评估的作用 在催化不涉及净氧化的酶中， 还原反应棒状杆菌肌氨酸氧化酶，一种有用的酶 在临床诊断肾功能方面，是一种异四聚体蛋白 （（alpha beta delta gamma）含有共价（beta subunit）和 非共价黄素，肌氨酸的单一结合位点和两个位点 另一种底物四氢叶酸β亚基表现出 与各种单体胺氧化还原酶的序列同源性， 只有共价黄素。为了确定这个新的家庭 黄素酶还包括哺乳动物肌氨酸脱氢酶，我们计划 对猪和人类肌氨酸脱氢酶的基因进行测序。信息 对人类基因的研究可能有助于阐明 肌氨酸血症是一种常染色体隐性遗传疾病， 肌氨酸脱氢酶缺乏， 血浆和尿液，并可能与神经和其他 问题复杂四级结构的功能意义 将在研究中探索棒状杆菌肌氨酸氧化酶， 各种配体的结合位点，并确定是否所有亚基 对肌氨酸氧化至关重要 我们亦希望澄清 电子从肌氨酸转移到非共价黄素的机制， 核黄素电子转移的动力学和热力学， 生物合成和共价黄素键的催化意义。 我们计划鉴定形成可逆复合物的半胱氨酸残基 与共价黄素结合， 酶的调节。人肌氨酸脱氢酶的作用机制， 包括它与四氢叶酸的相互作用， 生物合成的共价黄素连接，将进行研究， 与更复杂的棒状杆菌酶相比。另一个项目 涉及来自大肠杆菌和酵母的DNA光解酶 啤酒。这些酶含有蝶呤和还原黄素发色团 使它们能够利用可见光修复嘧啶二聚体， 由于DNA暴露在紫外线下而造成的严重损伤。如果放任 如果不修复，这种DNA损伤会导致突变、癌症和细胞死亡。 蝶呤发色团与N-末端结构域结合，并作为 天线，收集光能，然后转移到减少 黄素还原的黄素结合到C-末端结构域， 单重态是直接与底物相互作用的物质， 启动DNA修复我们试图确定酵母的结构 光裂合酶，并研究其特异性和重要性， 通过产生嵌合酶的结构域间相互作用。我们寻求 表征黄素环境和任何扰动引起的 衬底 我们试图研究二聚体修复的机制， 涉及时间分辨光谱技术的研究。