Mechanistic Studies of Flavoprotein Oxidases

黄素蛋白氧化酶的机理研究

• 批准号: 9506060
• 负责人: Paul Fitzpatrick
• 金额: $ 24.48万
• 依托单位: Texas A&M Research Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506060&HistoricalAwards=false
• 关键词: Mechanistic; Studies; Flavoprotein; Oxidases

; R o o t E n t r y F : @ C o m p O b j b W o r d D o c u m e n t # O b j e c t P o o l : : F Microsoft Word 6.0 Document MSWordDoc Word.Document.6 ; Oh +' 0 $ H l D h R:\WWUSER\TEMPLATE\NORMAL.DOT marcia steinberg marcia steinberg @ P: @ e = e # j j j j j j j O L # 1 T 4 O j O j j j j ~ j j j j 4 9506831 Plapp Alcohol dehydrogenases from horse liver and yeast have been studied extensively, but the origin of the catalytic activity and the function of the whole protein in catalytic dynamics is not fully understood. Roles for some amino acid residues at the active sites have been proposed from knowledge of the three dimensional structures, chemical modification, pH dependencies, and site directed mutagenesis, but contributions to catalysis should be quantitatively evaluated. Although enzymologists consider that residues at the active site are most critical for catalysis, emerging data suggest that amino acid residues distant from the active site contribute indirectly to catalytic power. Moreover, conformational change s can affect catalysis in unknown ways. Alcohol dehydrogenases offer a good system for studying these and other aspects of dynamics and catalysis. Three dimensional structures of the liver enzyme in two conformational states have been solved, and amino acid sequences for more than 60 alcohol dehydrogenases are available. Rate and dissociation constants for each step in the mechanism can be estimated from steady state and transient data and can quantitatively assess the effects of structural changes. The objectives of the proposed research are to answer questions about (1) the pH dependence of the catalytic mechanism, (2) the contribution of protein dynamics and conformational changes to catalysis and hydrogen tunneling, and (3) the tertiary and quaternary structures of various states of the horse and yeast enzymes. Site directed mutagenesis will be used to substitute amino acid residues, and steady state and transient kinetics, x ray crystallography, NMR, and fluorescence will be used to study these variants. Amino acid residues in the proton relay system and other residues at or distant from the active site will be substituted in order to study the origin of the pH dependencies for coenzyme binding and substrate interconversion steps. Residues near the coenzyme and substrate binding sites will be studied, as will some residues that contribute to the overall electrostatic potential of the protein. Protein flexibility and hydrogen tunneling will be studied in the liver enzyme with substitutions of residues in the interiors of the domains and at hinge regions, and by deletion of Q loops or other "extraneous" structural elements. Dynamic fluorescence properties of the horse liver enzyme with substituted tryptophan residues will be assessed. Three dimensional structures of complexes of the tetrameric yeast alcohol dehydrogenase and of potentially new conformational states of the dimeric horse liver enzyme will be determined by x ray crystallography. This research should increase our understanding of biocatalysis. %%% Alcohol dehydrogenase is the enzyme required for the production of ethanol by fermentation in yeast and for the removal of alcohols by metabolism in man. The enzyme accelerates the rate of dehydrogenation of alcohols with a coenzyme, but the catalytic mechanism is not yet fully understood. Enzymes are large proteins that have a smaller active site region with several amino acid residues that directly bind the alcohol and the coenzyme. Other amino acid residues outside of the active site provide a framework for the catalytic process and indirectly affect the rate of the reaction by controlling protein dynamics. Three dimensional structures of the horse and human liver enzymes have been determined, and now the structure of the yeast enzyme will be determined. The horse liver enzyme changes conformation during catalysis, which may facilitate the transfer of hydrogen by a "tunneling" process. The roles of the various amino acid residues in the horse and yeast enzymes will be studied by "protein engineering", which uses site directed mutagenesis to change individual amino acids, and by steady state and rapid reaction kinetics, X ray crystallography, and other spectroscopic methods that can detect the effects of the mutations. These studies will increase our understanding of biocatalysis. *** @ @ ; S u m m a r y I n f o r m a t i o n (

;​R o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o​F Microsoft Word 6.0文档MSWordDoc。6;Oh +' 0 $ H l D H R:\WWUSER\TEMPLATE\NORMAL。DOT marcia steinberg @ P：@ e = e # jjjjjjjjjjjjjjjj0 # 1 T 4 jjj0马肝和酵母中的Plapp醇脱氢酶已被广泛研究。但其催化活性的来源及整个蛋白在催化动力学中的作用尚不完全清楚。一些氨基酸残基在活性位点的作用已经从三维结构、化学修饰、pH依赖性和位点定向诱变等方面提出，但对催化的贡献还需要定量评估。尽管酶学家认为活性位点上的残基对催化作用最为关键，但新出现的数据表明，远离活性位点的氨基酸残基间接影响催化能力。此外，构象变化可以以未知的方式影响催化。醇脱氢酶为研究这些和其他方面的动力学和催化提供了一个很好的体系。肝酶在两种构象状态下的三维结构已经被解出，并获得了60多种醇脱氢酶的氨基酸序列。从稳态和瞬态数据中可以估计出机制中每个步骤的速率和解离常数，并可以定量评估结构变化的影响。提出的研究目标是回答以下问题：(1)催化机制的pH依赖性；(2)蛋白质动力学和构象变化对催化和氢隧道的贡献；(3)马和酵母酶的各种状态的三级和四级结构。位点定向诱变将用于替代氨基酸残基，稳态和瞬态动力学、x射线晶体学、核磁共振和荧光将用于研究这些变异。为了研究辅酶结合和底物相互转化步骤的pH依赖性的起源，将取代质子接力系统中的氨基酸残基和活性位点附近或远处的其他残基。将研究辅酶和底物结合位点附近的残基，以及对蛋白质的总静电电位有贡献的一些残基。蛋白质的柔韧性和氢隧道将在肝酶中通过取代结构域内部和铰链区域的残基，以及通过删除Q环或其他“外来”结构元件来研究。动态荧光性质的马肝酶取代色氨酸残基将进行评估。四聚体酵母醇脱氢酶配合物的三维结构和二聚体马肝酶潜在的新构象状态将用x射线晶体学测定。这项研究应该增加我们对生物催化的认识。酒精脱氢酶是酵母发酵生产乙醇和人体代谢去除酒精所必需的酶。。该酶与辅酶一起加速醇的脱氢速率，但其催化机制尚不完全清楚。酶是一种大型蛋白质，具有较小的活性位点区域，有几个氨基酸残基直接结合醇和辅酶。活性位点外的其他氨基酸残基为催化过程提供了框架，并通过控制蛋白质动力学间接影响反应速率。马和人肝酶的三维结构已经确定，现在酵母酶的结构将被确定。马肝酶在催化过程中改变构象，这可能通过“隧道”过程促进氢的转移。各种氨基酸残基在马和酵母酶中的作用将通过“蛋白质工程”来研究，它使用位点定向诱变来改变单个氨基酸，并通过稳态和快速反应动力学，X射线晶体学和其他可以检测突变影响的光谱方法来研究。这些研究将增加我们对生物催化的理解。*** @ @；如果我是你，我是你，我是你，我是你，我是你。