SPECIFIC ACTIVATORS OF LIVER ALCOHOL DEHYDROGENASE

这些研究的总体目标是更多地了解 马肝乙醇脱氢酶的机制，特别是关于 当辅酶结合时和当三元时发生的构象变化 复合物相互转化。 构象变化的速率将是 使用快速反应技术用各种光谱探针测定。 由于赖氨酸残基228的修饰增加了活性， 10倍，显然是通过改变酶变化的速率 构象，活化机制和速率， 将确定构象变化。 功能组的作用 在活性位点将阐明化学修饰和pH值 依赖性研究。 三元配合物相互转化的平衡位置 （“内部平衡”），这是衡量“效率”的标准。 酶将被确定。 pH依赖性，变化的影响 反应物的氧化还原电位，以及结合的倾向 “非生产性”模式的特点。 的尺寸和形状 衬底结合袋将相对于 蛋白质的三维结构。 基于对酶的结构和机制的了解， 将合成和评估活化剂。 一种类型的活化剂是 具有可使氨基甲基化的官能团的辅酶类似物 赖氨酸残基228。 第二种类型的激活剂是参与 在偶联交换反应中并氧化酶NADH复合物， 从而绕过辅酶的限速解离。 这些 活化剂应该有助于确定酒精的限速作用 脱氢酶在乙醇代谢，并最终用于治疗 急性和慢性酒精中毒。

The overall goal of these studies is to learn more about the detailed mechanism of horse liver alcohol dehydrogenase, in particular about conformational changes that occur when coenzymes bind and when ternary complexes interconvert. The rates of the conformational changes will be determined with various spectral probes using rapid reaction techniques. Since modification of lysine residue 228 increases the activity up to 10-fold, apparently by altering the rate at which enzyme changes conformation, the mechanism of the activation and the rates of the conformational change will be determined. The roles of functional groups at the active site will be elucidated by chemical modification and pH dependency studies. The equilibrium position for the interconversion of ternary complexes ("internal equilibrium"), which is a measure of the "efficiency" of the enzyme will be determined. The pH dependencies, the effects of variation of redox potential of the reactants, and the propensity to bind in "non-productive" modes will be characterized. The size and shape of the substrate binding pocket will be defined in relation to the three-dimensional structure of the protein. Based on knowledge of the structure and mechanism of the enzyme, specific activators will be synthesized and evaluated. One type of activator is a coenzyme analog with functional groups that can methylate the amino group of lysine residue 228. A second type of activator is one that participates in a coupled exchange reaction and oxidizes the enzyme NADH complex, thereby by-passing the rate-limiting dissociation of coenzyme. These activators should be useful for defining the rate-limiting role of alcohol dehydrogenase in ethanol metabolism and eventually for the treatment of acute and chronic alcoholism.