ENZYMOLOGY OF THE REDUCTIVE ACETYL-COA PATHWAY

还原乙酰辅酶途径的酶学

• 批准号: 6635972
• 负责人: Stephen Wiley Ragsdale
• 金额: $ 26.24万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6635972
• 关键词: Clostridium; acetyl coA; active sites; aldehyde /ketone oxidoreductase; carbon monoxide; chemical kinetics; cobamide; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; ferredoxin; iron sulfur protein; metalloenzyme; methyltransferase; microorganism metabolism; nickel; nuclear magnetic resonance spectroscopy; protein structure function; site directed mutagenesis; stereochemistry; tetrahydrofolates; zinc

DESCRIPTION: (Verbatim from the Applicant's Abstract) This proposal focuses on structure-function studies on the key enzymes in the Wood-Ljungdahl pathway. Understanding the mechanisms of these enzymes is significant to the areas of enzymology, bioinorganic chemistry and microbiology. This pathway is important to human biochemistry since acetogens and methanogens are components of the intestinal flora. Studies of this pathway are uncovering the principles by which metal centers in enzymes perform nucleophilic reactions and couple redox reactions to chemical catalysis. The Clostridium thermoaceticum methyl transferase is the ideal paradigm for the class of B12-dependent methyl transferases. An important objective is to increase the resolution of the atomic model of methyl transferase and determine the structure with both B12 and CH3-H4folate bound. X-ray diffraction, site-directed mutagenesis, and kinetic studies will be used to identify the proton transfer pathway that leads to activation of CH3-H4folate for nucleophilic attack by the corrinoid iron-sulfur protein. Coordination chemistry of the cobalt center has been proposed to play a controlling role in redox chemistry and catalysis of B12-dependent methyl transferases. It is planned to measure the extent to which the lower axial ligand controls catalysis and redox chemistry and to determine the atomic structure of the corrinoid iron-sulfur protein. Mechanistic studies on CO dehydrogenase/acetyl-CoA synthase will include: determining its crystal structure; elucidating how the CO dehydrogenase and acetyl-CoA synthase active sites communicate to coordinate electron and carbon flow; determining whether the precursors of the carbonyl, methyl, and acetyl groups of acetyl-CoA bind to the Ni or Fe components of Cluster A in acetyl-CoA synthase; determining the binding order of methyl, carbonyl, and CoA moieties to Cluster A; and developing a way to express recombinant CO dehydrogenase/acetyl-CoA synthase in an active form. The electronic structure of a substrate-derived hydroxyethyl-TPP radical that has been shown to be a catalytic intermediate in the mechanism of the C. thermoaceticum pyruvate:ferredoxin oxidoreductase will be elucidated. The hypothesis that CO2 is channeled from pyruvate:ferredoxin oxidoreductase to CO dehydrogenase will be tested. Studies will be focused on determining how CoA binding affects electron transfer and catalysis and if a CoA radical may be a catalytic intermediate.

描述：（逐字研究申请人的摘要）该提案的重点是 结构 - 函数研究对木 - 林区途径中的关键酶。 了解这些酶的机制对于 酶学，生物学化学和微生物学。这条路很重要 对于人类的生物化学 肠道菌群。该途径的研究正在揭示原理 哪个金属中心在酶中进行亲核反应和夫妇氧化还原 对化学催化的反应。 甲基甲基转移酶的梭状芽胞杆菌是理想的范式 B12依赖性的甲基转移酶。一个重要的目标是 增加甲基转移酶原子模型的分辨率并确定 具有B12和CH3-H4叶酸结合的结构。 X射线衍射， 定点诱变和动力学研究将用于确定 质子转移途径，导致CH3-H4叶酸激活 高核酸铁硫蛋白的亲核发作。协调 钴中心的化学反应已被提议在 B12依赖性甲基转移酶的氧化还原化学和催化。这是 计划测量较低轴向配体控制的程度 催化和氧化还原化学，并确定 抗性铁硫蛋白。关于CO的机械研究 脱氢酶/乙酰-COA合酶将包括：确定其晶体 结构;阐明CO脱氢酶和乙酰-COA合酶的活性 地点通信以协调电子和碳流；确定是否 乙酰辅酶A的羰基，甲基和乙酰基的前体与 乙酰-COA合酶中簇A的Ni或Fe成分；确定 甲基，羰基和COA部分的结合顺序以簇A；和 开发一种表达重组CO脱氢酶/乙酰-COA合酶的方法 活跃形式。底物衍生的电子结构 羟基乙基-TPP自由基已被证明是催化中间体 丙酮酸梭状芽胞杆菌的机制：铁氧还氧化还原酶将 阐明。二氧化碳是从丙酮酸引导的假设：铁氧还蛋白 将测试氧化氧化酶至CO脱氢酶。研究将集中在 确定COA结合如何影响电子传递和催化以及是否影响A COA自由基可能是催化中间体。