STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

乙醇脱氢酶的结构功能研究

• 批准号: 3109431
• 负责人: BRYCE V PLAPP
• 金额: $ 10.66万
• 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/3109431
• 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.

已经研究了马肝和酵母的酒精脱氢酶 广泛。 马肝酶的三维结构 并且已知一些具有底物和配体的复合物，以及基因 对于三种酵母菌酒精脱氢酶，已克隆在质粒中。 因此， 现在可以向有关该问题的几个杰出问题获得答案 酶的催化机制，动力学的相关性 结构和功能的特征，以及 活动中的第三和第四纪结构。 “特定地点 诱变将用于制备饮酒脱氢酶的变体 以下研究。 质子继电器系统的重要性 包括HIS-51和SER-48在肝酶中，将由 将这些残留物更改为无法参与质子的残留物 中继。 氨基酸残基（ASP-49和GLU-68），贡献羧基 将取代催化锌离子环境的组 残留无负荷。 底物装订口袋的大小将是 增加或减少以及对基材和速率的影响 将确定增强特异性。 酶的动力学 在生理条件下，体外将与体内的通量有关 以及酵母的增长率。 将尝试改变 酶对辅酶和底物的特异性，例如 进行替换，使酶可以将NADP作为辅酶结合 和l-lactate作为底物。 结构锌在活动中的作用 将通过去除结合锌的残基来检查。 残留物 假定的四二聚体之间的接触区域 将改变酶以制备二聚体酵母酶 肝脏形式。 分子的外循环或区域将是 删除是为了制造最小的催化单元。 酵母菌 脱氢酶将结晶以确定结构 X射线晶体学。 马肝酒脱氢酶的cDNA将 克隆相同类型的研究。 这项研究应该揭示 酒精结构和功能相关的基本方面 脱氢酶。 这样的基本研究是需要改善我们的 对生物催化的一般理解。 他们还应该帮助解释 该酶的生理功能。