FLAVOENZYME MECHANISMS--REDOX AND NONREDOX REACTIONS

我们的长期目标是阐明氧化的机制- 黄素酶催化还原反应及其作用的评价 在催化不涉及净氧化的酶中的黄素- 还原反应。棒状杆菌肌氨酸氧化酶，一种有用的酶 在临床诊断肾功能时，是一种异四聚体蛋白 (包含共价(β亚基)的(阿尔法-贝塔-伽马)和 非共价黄素，肌氨酸的一个结合部位和两个结合部位 对于另一种底物，四氢叶酸。(β亚基展示 含有多种单体胺氧化还原酶的序列同源性 只有共价黄素。以确定这个新的家族是否 黄素酶还包括哺乳动物的肌氨酸脱氢酶，我们计划 测定猪和人肌氨酸脱氢酶基因的序列。信息 对人类基因的研究可能有助于阐明人类免疫缺陷的分子基础 肌氨酸血症是一种常染色体隐性遗传病，其特征是 肌氨酸脱氢酶缺乏，肌氨酸水平升高 血浆和尿液，可能与神经系统和其他 有问题。复杂四元结构的功能意义 将在研究中探索棒状杆菌肌氨酸氧化酶的位置 各种配体的结合位点，并确定是否所有亚基 对肌氨酸氧化是必不可少的。我们还试图澄清 肌氨酸向非共价黄素的电子转移机制， 黄素间电子转移的动力学和热力学，以及 黄素共价键的生物合成及其催化意义。 我们计划鉴定形成可逆复合体的半胱氨酸残基 与黄素的共价键似乎代表了一种新的机制 酶的调节作用。人肌氨酸脱氢酶的作用机制， 包括其与四氢叶酸的相互作用及其作用 将研究共价黄素键的生物合成，并 与更复杂的棒状杆菌酶相比。另一个项目 涉及大肠杆菌和酵母菌的DNA光解酶 酿酒。这些酶含有蝶呤蛋白和还原的黄素发色团。 使它们能够用可见光修复嘧啶二聚体， DNA暴露在紫外光下造成的主要损害。如果离开 如果不能修复，这种DNA损伤可能会导致突变、癌症和细胞死亡。 蝶呤蛋白发色团结合到N-末端结构域，并作为 天线，收集光能，然后转移到还原的 弗拉文。还原的黄素与C-末端结构域结合并被激发 单线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 光解酶，并研究其特异性和重要性 通过产生嵌合酶进行域间相互作用。我们寻求 描述Flavin环境和由其引起的任何扰动 底物。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。

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.