MECHANISMS INVOLVED IN FLAVIN-LINKED OXYGEN METABOLISM

黄素连接氧代谢的机制

• 批准号: 3288058
• 负责人: AL CLAIBORNE
• 金额: $ 10.46万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288058
• 关键词: Enterococcus; atomic absorption spectrometry; circular dichroism; dithiol; enzyme complex; enzyme mechanism; enzyme structure; enzyme substrate; flavin adenine dinucleotide; flavoproteins; genetic library; genetic manipulation; glycerol 3 phosphate dehydrogenase; peroxidases; stoichiometry; stop flow technique

The long-term objectives of this study are to understand the mechanisms by which three unique streptococcal flavoproteins catalyze reactions normally ascribed to hemeproteins or to heme- dependent processes. Investigation of the catalytic roles of both flavin and non-flavin redox centers will be essential to the overall evaluation of the key structure-function relationships operative in the three enzymes, which are important in streptococcal oxygen metabolism. Recombinant DNA approaches will be applied to the study of the FAD- containing NADH peroxidase: the specific questions to be addressed include the cysteinyl peptide sequences of the proposed redox- active disulfide and the overall relatedness between the peroxidase and the flavoprotein disulfide reductases. In the context of our mechanistic studies, the roles of two distinct complexes of the reduced peroxidase with pyridine nucleotides in both peroxide reduction and in transhydrogenation will be investigated by static titration and rapid-reaction techniques, as will the specific role of cysteine thiol(s) in peroxide reduction. Studies of the unusual FAD-containing NADH oxidase will focus on the identification of the second non-flavin redox center in this enzyme and on the interaction of this center with the flavin in the overall four-electron reduction of oxygen. The possible role of oxygenated flavin intermediates in the catalytic mechanism will also be investigated by stopped-flow methods and will include studies of the enzyme reconstituted with modified FAD analogs. The possible structural and functional relationship of the NADH oxidase and the NADH peroxidase will be examined by protein chemistry and molecular genetic approaches. There are two major questions concerning the streptococcal alpha- glycerophosphate oxidase. The distinction between hydride- and carbanion-mediated substrate dehydrogenations is an important one, and halogenated analogs of the natural substrate will be used to probe this question in more detail. Secondly, the comparison between this flavoprotein and the FAD-containing anaerobic alpha- glycerophosphate dehydrogenase of E. coli should provide valuable information concerning structural and mechanistic features shared by both.

这项研究的长期目标是了解 三种独特的链球菌黄蛋蛋白的机制 催化通常归因于血蛋白或血红素的反应 依赖过程。 研究两者的催化作用 黄素和非流行氧化还原中心对于 关键结构功能关系的总体评估 在三种酶中的手术者，这在 链球菌氧代谢。 重组DNA方法将应用于FAD-的研究 含有NADH过氧化物酶：要解决的具体问题 包括所提出的氧化还原的半胱氨酸肽序列 活性二硫化物和过氧化物酶之间的总体相关性 以及黄蛋白二硫化物还原酶。 在我们的背景下 机械研究，两个不同复合物的作用 两种过氧化物中用吡啶核苷酸的过氧化物酶还原过氧酶 降低和经氢化中的静电将通过静态研究 滴定和快速反应技术，具体角色也 在过氧化物还原中的半胱氨酸硫醇。 对含有FAD FAD的NADH氧化酶的研究将集中在 在此识别第二个非流但氧化还原中心 酶以及该中心与黄素的相互作用 总体四电子还原氧。 可能的作用 催化机制中的氧化黄素中间体将 也可以通过停止流量的方法进行调查，并将包括 对酶的研究用改良的FAD类似物重构。 这 NADH氧化酶可能的结构和功能关系 NADH过氧化物酶将通过蛋白质化学和 分子遗传方法。 关于链球菌α-有两个主要问题 甘油磷酸氧化酶。 氢化物和 Carbanion介导的底物脱氢是一个重要的脱氢作用， 自然基材的卤代类似物将用于 更详细地探究这个问题。 其次，比较 在这种黄素蛋白和含有FAD的厌氧α之间 大肠杆菌的甘油磷酸脱氢酶应提供有价值的 有关共享结构和机械特征的信息 两者。