STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

乙醇脱氢酶的结构功能研究

• 批准号: 3109435
• 负责人: BRYCE V PLAPP
• 金额: $ 20.32万
• 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/3109435
• 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 biosynthesis; 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.

从马肝和酵母中提取的乙醇脱氢酶已经被 广泛研究。 的三维结构， 马肝酶和几种与底物的复合物， 配体是已知的，四种酵母醇的基因 已在质粒中克隆了内切酶。 因此，答案可以 现在可以得到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， 三级和四级结构参与活动。 “位点特异性诱变”将用于制备 乙醇脱氢酶用于以下研究。 的重要性 质子中继系统，其中包括His-51和Ser-48， 将通过改变这些残留物来研究肝酶 到那些不能参加质子接力的人。 氨基酸 有助于催化锌环境的残留物 离子将被取代，辅酶中涉及的氨基酸也将被取代 约束力 基底结合袋的尺寸将是 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 在体外生理条件下的酶将与 体内的通量和酵母的生长速率。 将尝试 改变酶对辅酶的特异性 和底物，例如，进行取代， 允许酶结合NADP作为辅酶和L-乳酸作为辅酶， 衬底 结构锌在活性中的作用将是 通过去除结合锌的残留物进行检查。 残留 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 一种二聚酵母酶，类似于肝脏形式。 外源性循环或 分子的某些区域将被移除， 最小催化单位 酵母醇脱氢酶将是 结晶，用于通过X射线确定结构 结晶学 马肝乙醇脱氢酶cDNA 将被克隆用于相同的研究。