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: 2176271
• 负责人: MARILYN S JORNS
• 金额: $ 24.58万
• 依托单位: ALLEGHENY UNIVERSITY OF HEALTH SCIENCES
• 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 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176271
• 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 - 减少黄酮酶催化的反应并评估该作用 催化不涉及净氧化的酶中黄素的黄素 还原反应。 Corynebacterial Sarcosine氧化酶，一种有用的酶 在肾功能的临床诊断中，是一种异次术蛋白 （（（alpha beta delta伽玛）包含共价（beta子单位）和 非共价黄素，肌苷的单个结合位点和两个位点 对于另一个底物四氢叶酸。 （beta亚基展览品 与含有各种单体胺氧化还原酶的序列同源性 只有共价黄素。确定这个新家庭是否 黄酮酶还包括哺乳动物肌动脉脱氢酶，我们计划 序列猪和人肌苷脱氢酶的基因。信息 在人类基因上可能有用 Sarcosinemia是一种常染色体隐性疾病，其特征是 肌苷脱氢酶的缺乏和高水平的肌苷在 血浆和尿液，可能与神经系统和其他 问题。复杂四级结构的功能意义 在研究中将探测Corynebacterial Sarcosine氧化酶的定位 各种配体的结合位点，并确定是否所有亚基 对于肌苷氧化至关重要。 我们还试图阐明 电子从肌苷转移到非共价黄素的机制， Flavin电子转移的动力学和热力学，以及 共价黄素连接的生物合成和催化意义。 我们计划识别形成可逆复合物的半胱氨酸残基 用共价黄素代表了一种新型机制 酶调节。人肌别脱氢酶的机制， 包括其与四氢叶酸的相互作用以及角色和 共价黄素连锁的生物合成将被研究，并 与更复杂的corynybacterial酶相比。另一个项目 涉及来自Escherichia卷曲和糖疗法的DNA光溶酶 酿酒酵母。这些酶含有翼龙和减少黄素发色团 这使他们能够使用可见光修复嘧啶二聚体 DNA暴露于紫外线引起的重大损害。如果离开 该DNA损伤未经修复会导致突变，癌症和细胞死亡。 翼蛋白发色团与N末端结构域结合，并充当 天线，收获光能，然后转移到还原 黄素。减少黄素结合了C末端结构域，其激发 单线状态是直接与底物相互作用的物种， 启动DNA修复。我们试图确定酵母的结构 光溶解酶并研究的特异性和重要性 通过创建嵌合酶进行域间相互作用。我们寻求 表征黄素环境和任何造成的扰动 基材。 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。