SPECIFIC ACTIVATORS OF LIVER ALCOHOL DEHYDROGENASE

这些研究的总体目标是更多地了解 马肝醇脱氢酶的作用机制，特别是关于 辅酶结合和三元结合时发生的构象变化 复合体相互转化。构象变化的速率将是 通过使用快速反应技术的各种光谱探针进行测定。 由于对赖氨酸残基228的修饰将活性提高到 10倍，显然是通过改变酶的变化速度 酶的构象、活化机制和速率 构象变化将被确定。官能团的作用 活性中心的位置将通过化学修饰和pH来确定 依赖性研究。 三元络合物相互转化的平衡位置 (“内部均衡”)，这是一个衡量“效率”的指标 酶将会被测定。酸碱度的依赖关系，变化的影响 反应物的氧化还原电势和结合的倾向 将以“非生产性”模式为特征。的大小和形状 衬底绑定口袋将根据以下内容定义 蛋白质的三维结构。 基于对酶的结构和机制的知识，特定的 将对激活剂进行合成和评估。一种类型的激活剂是 具有可甲基化氨基的官能团的辅酶类似物 有228个赖氨酸残基。第二种类型的激活剂是一种参与 在耦合交换反应中并氧化酶NADH复合体， 从而绕过辅酶的限速解离。这些 激活剂对于确定酒精的限速作用是有用的 脱氢酶在乙醇代谢中的作用，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.