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-水溶液和零度以下的真实反应温度 肝醇脱氢酶中间体可以稳定和 具有结构特征的。发色醇的合成 4-(2-咪唑基)-偶氮苯醇被描述为用作直接动力学 低温酶学研究中乙醇氧化问题的探讨。这种合成物 基质在近处应表现出中等强度的吸收 紫外区(350-400 nm)，与 Coenyzme NAD+。一旦在以下基础上确定了冷冻溶剂条件 稳定催化活性的低温动力学实验 酶-NAD+天然酶的乙醇复合体，适用于 活性部位特异的钴离子重组酶用于结构 活性中心金属配位环境的表征 离子的电子顺磁共振方法。符合以下条件的 稳定反应中间体也将被应用于确定 催化活性酶NAD+的底物构型 酒精情结。为此，自旋标记物的合成 氮氧化物衬底3-(2，2，5，5-tetra-methylpyrrolinyl-1-oxyl)-propen-1-o1 被描述为用作光谱探测器。使用Co++ 重组酶，金属离子与氮氧化物的距离 负温稳定的酶-NAD+乙醇底物复合体 可以根据激冷的偶极相互作用确定 氮氧化物部分的EPR信号幅度。这种数量结构 然后可以使用参数来搜索立体化学相容 活性中心底物构型的分子图形研究 技巧。结构环境的直接确定 催化活性反应中间体中的活性中心金属离子 将能够指定醇氧化的结构基础 由肝醇脱氢酶催化。

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.