STRUCTURE-FUNCTION STUDIES OF ALCOHOL DEHYDROGENASES

马肝脏和酵母中的酒精脱氢酶已被 经过广泛的研究。的三维结构 马肝酶及其几种底物和几种络合物 配基是已知的，四种酵母酒精的基因 脱氢酶已被克隆到质粒中。因此，答案可以 现在得到了几个关于 酶的催化机理、动力学关联性 结构和功能的特点，以及 三、四级结构参与活动。 “定点突变”将被用来制备变种的 酒精脱氢酶用于以下研究。它的重要性 包括His-51和Ser-48在内的质子中继系统 肝酶，将通过改变这些残留物来研究 那些不能参加质子接力的人。氨基酸 对催化锌的环境有贡献的残留物 离子将被取代，参与辅酶的氨基酸也将被取代 有约束力的。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。该反应的动力学 酶在体外的生理条件下将与 体内的通量和酵母的生长速度。一次尝试将 改变酶对辅酶的专一性 和底物，例如，通过进行替换来实现 允许酶以辅酶形式结合二磷酸腺苷，以辅酶形式结合L-乳酸 底物。结构锌在活性中的作用将是 通过去除束缚锌的残留物进行检查。残留物在 分子的两个二聚体之间的假设接触区域 四聚体酵母酶将被改变，试图准备 一种二聚体酵母酶，类似于肝脏形式。无关的循环或 分子的区域将被移除，以尝试制造 最小催化单位。酵母醇脱氢酶将是 结晶，用于x射线结构测定 结晶学。马肝酒精脱氢酶基因的克隆 将被克隆用于相同类型的研究。

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.