STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

乙醇脱氢酶的结构功能研究

• 批准号: 3109433
• 负责人: BRYCE V PLAPP
• 金额: $ 18.42万
• 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 1991-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3109433
• 关键词: Schizosaccharomyces pombe; X ray crystallography; affinity chromatography; alcohol dehydrogenase; catalyst; chemical structure function; cofactor; complementary DNA; conformation; dimer; enzyme mechanism; enzyme structure; enzyme substrate; gel electrophoresis; high performance liquid chromatography; horses; hydrogen transport; isozymes; liver cells; molecular cloning; molecular site; mutant; nucleic acid sequence; protein engineering; protein 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 four 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 that contribute to the environment of the catalytic zinc ion will be substituted, as will amino acids involved in coenzyme binding. 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.

马肝和酵母的酒精脱氢酶已经 广泛研究。 三维结构 马肝酶和几种带有底物的复合物， 配体是已知的，四个酵母菌的基因 脱氢酶已被克隆在质粒中。 因此，答案可以 现在可以解决有关 酶的催化机制，动力学的相关性 结构和功能的特征，以及 第三和第四纪结构参与活动。 “位点特异性诱变”将用于制备变体 酒精脱氢酶用于以下研究。 重要性 质子继电器系统，其中包括His-51和Ser-48 将通过更改这些残基来研究肝酶 对于不能参与质子继电器的人。 氨基酸 有助于催化锌环境的残留物 离子将被取代，与辅酶有关的氨基酸也将被取代。 结合。 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。 动力学 体外生理条件下的酶将与 体内的通量和酵母的增长率。 尝试 使酶对辅酶的特异性进行更改 例如，通过替换将 允许酶将NADP作为辅酶结合，而L乳酸作为A 基材。 结构锌在活动中的作用将是 通过去除结合锌的残基检查。 残留物 假定的接触区域之间的两个二聚体 四聚酵母酶将被改变以进行准备 像肝脏形式一样的二聚体酵母酶。 外循环或 分子的区域将被删除，以试图做一个 最小催化单元。 酵母酒精脱氢酶将是 结晶以确定X射线结构 晶体学。 马肝脱氢酶的cDNA 将被克隆用于相同类型的研究。