STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

乙醇脱氢酶的结构功能研究

• 批准号: 3109427
• 负责人: BRYCE V PLAPP
• 金额: $ 18.48万
• 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/3109427
• 关键词: Schizosaccharomyces pombe; X ray crystallography; affinity chromatography; alcohol dehydrogenase; catalyst; chemical structure function; cofactor; 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 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.

来自马肝和酵母的乙醇脱氢酶 广泛研究。 的三维结构 马肝酶和几种底物复合物 配体是已知的，四种酵母醇的基因 脱氢酶已被克隆到质粒中。 因此，答案可以 现在可以得到关于的几个悬而未决的问题 酶的催化机理、动力学相关性 结构和功能的特点，以及 三级和四级结构参与活动。 “位点特异性诱变”将用于制备 醇脱氢酶用于以下研究。 重要性 质子中继系统，其中包括 His-51 和 Ser-48 肝酶，将通过改变这些残留物进行研究 那些不能参加质子传递的人。 氨基酸 对催化锌环境有贡献的残留物 离子将被取代，辅酶中涉及的氨基酸也将被取代 绑定。 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。 的动力学 体外生理条件下的酶与 体内通量和酵母的生长速率。 一次尝试将 改变酶对辅酶的特异性 和底物，例如，进行替换 让酶结合 NADP 作为辅酶和 L-乳酸作为辅酶 基材。 结构锌在活性中的作用是 通过去除与锌结合的残留物进行检查。 残留物在 两个二聚体之间的假定接触区域 四聚体酵母酶将被改变以试图制备 一种像肝脏形式的二聚体酵母酶。 无关的循环或 分子的区域将被去除以试图制造 最小催化单元。 酵母乙醇脱氢酶将是 结晶以通过 X 射线确定结构 晶体学。 马肝乙醇脱氢酶的cDNA 将被克隆用于同类研究。