COOPERATIVITY IN ALCOHOL DEHYDROGENASE

该项目的总体目标是确定 肝脏酒精脱氢酶中的协同作用。肝醇 脱氢酶是乙醇代谢的限速因子， 强调其在消除乙醇方面的重要性。马与人 肝醇脱氢酶以同源和同源的混合物形式存在 异二聚体同工酶。已观察到明显的协作性 许多这种同工酶，特别是人类的Gamma2和Gamma2的同工酶 马和人肝脏的异二聚体同工酶 纸巾。重组人的稳态和暂态动力学 将测定人类的Gamma2-Gamma2酶，以努力了解 观察到的协作性。在这些研究的同时， 将测量伽马2-伽马-2人体酶的底物和辅酶 采用电子顺磁共振和荧光光谱分析。定点突变将 被用来破坏辅酶结合以确定构象是否 与与Gamma2Gamma2结合的辅酶相关的变化参与 调节亚单位间的相互作用。Alphagamma2的协作性 人肝同工酶和马肝异二聚体 野生型和突变型亚基也将被研究。结果是 这项研究将有助于目前对乙醇的理解 人类的新陈代谢。

The overall goal of this project is to determine the role of cooperativity in liver alcohol dehydrogenase. Liver alcohol dehydrogenase is a rate-limiting factor in ethanol metabolism, underscoring its importance in ethanol elimination. Horse and human liver alcohol dehydrogenases exist as a mixture of homo-and heterodimeric isoenzymes. Apparent cooperativity has been observed in many of these isoenzymes, specifically for the gamma2gamma2 human enzyme and the heterodimeric isoenzymes from both horse and human liver tissues. The steady-state and transient kinetics of recombinant gamma2gamma2 human enzyme will be determined in an effort to understand the observed cooperativity. Along with these studies, the binding of substrates and coenzymes to gamma2gamma2 human enzyme will be measured using epr and fluorescence spectroscopy. Site-directed mutagenesis will be used to disrupt coenzyme binding to determine if the conformational change associated with coenzyme binding to gamma2gamma2 is involved in mediating the subunit interactions. Cooperativity in the alphagamma2 human liver isoenzyme and in horse liver heterodimers consisting of a wild-type and a mutant subunit will also be studied. The results of this research will contribute to the current understanding of ethanol metabolism in man.