STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

乙醇脱氢酶的结构功能研究

• 批准号: 3109432
• 负责人: BRYCE V PLAPP
• 金额: $ 11.39万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-12-01 至 1986-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3109432
• 关键词: Schizosaccharomyces pombe; affinity chromatography; alcohol dehydrogenase; catalyst; chemical structure function; complementary DNA; conformation; dimer; enzyme structure; enzyme substrate; gel electrophoresis; high performance liquid chromatography; hydrogen transport; isozymes; liver cells; molecular cloning; molecular site; mutant; nucleic acid sequence; zinc

Alcohol dehydrogenases from horse liver and yeast have been studied extensively. The three-dimensional structures of the horse liver enzyme and several complexes with substrates and ligands are known, and the genes for three yeast alcohol dehydrogenases have been cloned in plasmids. Thus, answers can now be obtained to several outstanding questions about the catalytic mechanism of the enzyme, the correlation of kinetic characteristics with the structure and function, and the involvement of the tertiary and quaternary structures in activity. "Site-specific mutagenesis' will be used to prepare variants of alcohol dehydrogenases for the following studies. The importance of the proton relay system, which includes His-51 and Ser-48 in the liver enzyme, will be investigated by changing these residues to ones that cannot participate in the proton relay. Amino acid residues (Asp-49 and Glu-68) that contribute carboxyl groups to the environment of the catalytic zinc ion will be substituted with uncharged residues. The size of the substrate binding pocket will be increased or decreased and the effects on the substrate and rate enhancement specificity will be determined. The kinetics of the enzymes under physiological conditions in vitro will be related to the flux in vivo and to the growth rates of yeast. An attempt will be made to change the specificity of the enzyme for coenzyme and substrate by, for instance, making substitutions that will allow the enzyme to bind NADP as a coenzyme and L-lactate as a substrate. The role of the structural zinc in activity will be examined by removing residues that bind the zinc. Residues in the postulated contact regions between two dimers of the tetrameric yeast enzyme will be altered in an attempt to prepare a dimeric yeast enzyme like the liver form. Extraneous loops or regions of the molecule will be removed in an attempt to make a minimal catalytic unit. A yeast alcohol dehydrogenase will be crystallized for determination of the structure by x-ray crystallography. The cDNA for horse liver alcohol dehydrogenase will be cloned for the same kinds of studies. This research should reveal essential aspects of the correlation of structure and function in alcohol dehydrogenases. Such fundamental studies are required for improving our general understanding of biocatalysis. They should also help to explain the physiological functioning of this enzyme.

研究了来自马肝和酵母的乙醇脱氢酶 广泛地。 马肝酶的三维结构 已知几种具有底物和配体的复合物，并且基因 三种酵母醇脱氢酶已被克隆到质粒中。 因此， 现在可以获得有关该项目的几个悬而未决问题的答案 酶的催化机理、动力学相关性 结构和功能的特点以及参与 活性中的三级和四级结构。 “具体站点 “诱变”将用于制备醇脱氢酶的变体 以下研究。 质子中继系统的重要性 肝酶中包含 His-51 和 Ser-48，将通过以下方法进行研究 将这些残基改变为不能参与质子反应的残基 中继。 提供羧基的氨基酸残基（Asp-49 和 Glu-68） 催化锌离子环境中的基团将被取代 具有不带电的残留物。 基材结合袋的尺寸为 增加或减少以及对底物和速率的影响 将确定增强特异性。 酶的动力学 体外生理条件下与体内的通量有关 以及酵母的生长速度。 将尝试改变 酶对辅酶和底物的特异性，例如， 进行取代，使酶能够作为辅酶结合 NADP 和L-乳酸作为底物。 结构锌在活性中的作用 将通过去除与锌结合的残留物进行检查。 残留物在 四聚体酵母的两个二聚体之间的假设接触区域 酶将被改变以试图制备二聚体酵母酶，例如 肝脏的形式。 分子的无关环或区域将是 移除以试图制造最小的催化单元。 一种酵母酒精 脱氢酶将被结晶以测定结构 X 射线晶体学。 马肝乙醇脱氢酶的 cDNA 将 可以被克隆用于同类研究。 这项研究应该揭示 酒精结构与功能相关性的重要方面 脱氢酶。 需要进行此类基础研究来改善我们的 对生物催化的一般理解。 他们还应该帮助解释 这种酶的生理功能。