Rawat U B, Rao M B
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Eur J Biochem. 1997 Jun 1;246(2):344-9. doi: 10.1111/j.1432-1033.1997.00344.x.
Conformation and microenvironment at the active site of xylose reductase (XR) from Neurospora crassa was probed with fluorescent chemoaffinity labeling (FCAL) using o-phthalaldehyde as a chemical initiator. Formation of a single isoindole derivative resulted in complete inactivation of the enzyme as judged by spectroscopic and fluorescence studies. Kinetic analysis of the 2,4,6-trinitrobenzenesulfonic-acid-modified XR implicated the presence of an essential lysine residue at the active site of XR. Modification of lysine in XR abolished the ability of the enzyme to form isoindole derivative, indicating that the lysine residue involved in the reaction with 2,4,6-trinitrobenzenesulfonic acid and o-phthalaldehyde is the same and that the probe o-phthalaldehyde is directed to the active site. Fluorescence studies revealed that inactivation of XR by Gdn/HCl precedes gross conformational change and the possibility of secondary-conformational change was eliminated by acrylamide quenching studies. The enzyme inactivated by low concentrations of Gdn/HCl retained its ability to form the fluorescent XR-isoindole derivative indicating that inactivation is not due to conformational changes at or near the active site of XR. Gdn/HCl also had no effect on the high-affinity and low-affinity NADPH-binding sites of XR. Energy-transfer experiments further revealed structural integrity at the active site of the Gdn/HCl-inactivated XR. Changes in the fluorescence emission maximum of 1-(beta-hydroxyethylthio)-2-beta hydroxyethyl isoindole (EA adduct) in solvents of varying polarity was studied, the data obtained were utilized to interpret the fluorescence behaviour of XR-isoindole derivative and assess the polarity at the active site. Experimental evidence presented here serves to suggest that the inactivation of XR by Gdn/HCl precedes conformational changes at the active site located in a microenvironment of low polarity.
使用邻苯二甲醛作为化学引发剂，通过荧光化学亲和标记（FCAL）对粗糙脉孢菌木糖还原酶（XR）活性位点的构象和微环境进行了探究。光谱和荧光研究表明，单一异吲哚衍生物的形成导致该酶完全失活。对2,4,6-三硝基苯磺酸修饰的XR进行动力学分析，表明XR活性位点存在一个必需的赖氨酸残基。XR中赖氨酸的修饰消除了该酶形成异吲哚衍生物的能力，这表明与2,4,6-三硝基苯磺酸和邻苯二甲醛反应的赖氨酸残基是相同的，并且探针邻苯二甲醛指向活性位点。荧光研究表明，盐酸胍（Gdn/HCl）使XR失活先于其整体构象变化，并且通过丙烯酰胺猝灭研究排除了二级构象变化的可能性。低浓度Gdn/HCl使酶失活后，其仍保留形成荧光XR-异吲哚衍生物的能力，这表明失活并非由于XR活性位点或其附近的构象变化。Gdn/HCl对XR的高亲和力和低亲和力NADPH结合位点也没有影响。能量转移实验进一步揭示了Gdn/HCl失活的XR活性位点的结构完整性。研究了1-(β-羟乙硫基)-2-β-羟乙基异吲哚（EA加合物）在不同极性溶剂中荧光发射最大值的变化，利用所得数据解释XR-异吲哚衍生物的荧光行为并评估活性位点的极性。此处提供的实验证据表明，Gdn/HCl使XR失活先于位于低极性微环境中的活性位点的构象变化。
