THE OXIDATION OF ALCOHOL BY LIVER ALCOHOL DEHYDROGENASE

描述了旨在确定混合的条件的研究。 有机水溶剂和零度以下的温度下，真正的反应 可以稳定肝醇脱氢酶的中间体， 结构特征。 发色醇的合成 4-（2-咪唑基）-偶氮苄醇被描述为用作直接动力学反应， 低温酶学研究中乙醇氧化的探针。 这种合成 基质应在近红外波段表现出中等强度的吸收， 紫外（350-400 nm）区域，其不与 coenyzme NAD+。 一旦根据以下条件确定了低温溶剂条件， 稳定催化活性物质的低温动力学实验 酶-NAD+天然酶的醇复合物，它们应被应用于 活性位点特异性Co++重构酶 活性中心金属的配位环境的表征 离子的电子顺磁共振方法。 的条件 稳定反应中间体也将用于确定 催化活性酶-NAD+中底物的构型 酒精复合体 为此，自旋标记的合成 氮氧化物底物3-（2，2，5，5-四甲基吡咯啉基-1-氧基）-丙烯-1-醇 被描述用作光谱探针。 使用Co++ 重组酶中的金属离子与氮氧距离 在零度以下温度下稳定的酶-NAD+醇底物复合物 可以根据猝灭的偶极相互作用来确定 氮氧部分的EPR信号幅度。 这一数量结构 然后可以使用参数来搜索立体化学相容的 通过分子图形学研究底物在活性部位的构型 技术. 直接确定构造环境 催化活性反应中间体中的活性中心金属离子 将能够分配醇氧化的结构基础 由肝脏乙醇脱氢酶催化。

Studies are described that are designed to identify conditions of mixed organic-aqueous solvents and subzero temperatures under which true reaction intermediates of liver alcohol dehydrogenase can be stabilized and structurally characterized. Synthesis of the chromophoric alcohol 4-(2-imidazolyl)-azobenzylalcohol is described for use as a direct kinetic probe of alcohol oxidation in cryoenzymologic studies. This synthetic substrate should exhibit moderately intense absorption in the near ultraviolet (350-400 nm) region that is non-overlapping with that of the coenyzme NAD+. Once cryosolvent conditions are identified on the basis of cryokinetic experiments for stabilization of the catalytically competent enzyme-NAD+ alcohol complex of the native enzyme, they shall be applied to the active site specific Co++ reconstituted enzyme for structural characterization of the coordination environment of the active site metal ion by electron paramagnetic resonance methods. The conditions for stabilizing reaction intermediates will be applied also to determine the configuration of the substrate in the catalytically competent enzyme-NAD+ alcohol complex. For this purpose, the synthesis of the spin-label nitroxide substrate 3-(2,2,5,5-tetra-methylpyrrolinyl-1-oxyl)-propen-1-o1 is described for use as a spectroscopic probe. With use of the Co++ reconstituted enzyme, the metal ion-to-nitroxide distance in the enzyme-NAD+ alcohol substrate complex stabilized at subzero temperatures can be determined on the basis of the dipolar interactions that quench the EPR signal amplitude of the nitroxide moiety. This quantitative structural parameter can be then employed to search out stereochemically compatible configurations of the substrate in the active site by molecular graphics techniques. Direct determination of the structural environment of the active site metal ion in catalytically competent reaction intermediates will enable assignment of the structural basis of alcohol oxidation catalyzed by liver alcohol dehydrogenase.