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.

从马肝和酵母中提取的乙醇脱氢酶进行了研究。 广泛地。马肝酶的三维结构 以及已知的几种底物和配体的络合物，以及这些基因 对于三种酵母醇脱氢酶，都已克隆到载体中。因此， 现在可以得到关于 酶的催化机理、动力学关联性 具有结构和功能的特点，以及 活动中的三级和四级结构。“特定于站点 突变将被用于制备酒精脱氢酶的变种 进行了以下研究。质子接力系统的重要性，它 包括肝酶中的His-51和Ser-48，将由 将这些残基改变为不能参与质子的残基 接力赛。贡献羧基的氨基酸残基(Asp-49和Glu-68) 环境基团对锌离子的催化作用将被取代 含有不带电荷的残留物。衬底装订袋的大小为 增加或减少以及对底物和速率的影响 将确定增强的特异性。酶的动力学 在生理条件下，体外会与体内的流量有关 以及酵母的生长速度。将尝试更改 酶对辅酶和底物的专一性，例如， 进行替换，使酶能以辅酶的形式与NADP结合 以L-乳酸为底物。结构锌在活性中的作用 将通过去除结合锌的残留物进行检查。食品中的残留物 四聚体酵母两个二聚体之间的假设接触区域 酶将被改变以尝试制备二聚体酵母酶，如 肝脏的形态。分子的无关环或区域将是 在尝试制造最小催化单元时被移除。一种酵母酒精 脱氢酶将被结晶，以确定其结构 X射线结晶学。马肝酒精脱氢酶基因的克隆 被克隆用于相同类型的研究。这项研究应该会揭示 酒精结构与功能相互关系的基本问题 脱氢酶。这样的基础性研究是改善我们的 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。