COOPERATIVITY IN ALCOHOL DEHYDROGENASE

本项目的总体目标是确定 肝脏乙醇脱氢酶的协同性。 肝醇 脱氢酶是乙醇代谢中的限速因子， 强调了其在消除乙醇中的重要性。 马和人 肝乙醇脱氢酶以同型和同型混合物的形式存在， 异二聚体同工酶 表观协同性已被观察到， 这些同工酶中的许多，特别是人类的γ 2 γ 2酶 马和人肝脏的异二聚体同工酶 组织中 重组蛋白的稳态和瞬态动力学 将测定人γ 2 γ 2酶，以了解 观察到的协同性。 沿着这些研究， 将测量γ 2 γ 2人酶的底物和辅酶 使用EPR和荧光光谱法。 定点诱变将 用于破坏辅酶结合，以确定构象 与辅酶结合γ 2 γ 2相关的变化涉及 介导亚基相互作用。宇宙射线中的协同性2 人肝同工酶和马肝异源二聚体， 也将研究野生型和突变亚基。 的结果 这项研究将有助于目前对乙醇的理解 人体新陈代谢

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.