Determination of fine structure of the molybdo-enzyme and mechanism of hydroxylation and protein vibration.

我们一直在研究黄嘌呤氧化还原酶的构象变化和蛋白质结构的振动运动，这可能会产生活性氧。主要结果如下：1）通过X射线晶体学分析，确定了黄嘌呤氧化还原酶催化中心MoCo中心钼原子的配体及其几何构型。与以前的报道相反，对磺基和双磺基形式的电子密度的比较表明，氧原子位于顶端位置，而磺基原子位于赤道位置。这些结果解释了在FYX-051与慢底物的反应中间体结构中提出的羟基化反应过程中的氢化物转移机制。我们还提出的作用，位于活性位点空腔的氨基酸残基的嘌呤底物的羟基化的定点突变实验。2)我们通过三维结构分析揭示了脱氢酶转化为氧化酶的整体机制，并发表了特邀综述论文。3)为了解释在抗痛风药物中观察到的抑制剂对哺乳动物和细菌酶的不同影响，我们通过计算机模拟分析确定了分子动力学。结果表明，细菌和哺乳动物的酶在空腔中存在的疏水肽的动力学显着不同。4)我们已经构建了转基因小鼠，产生大量的超氧化物相比，正常的酶。表型分析仍在进行中。我们分析了黄嘌呤氧化还原酶抑制剂对ALS模型小鼠的作用，发现一些抑制剂对治疗应用相当有效。

We have been working on the conformational changes and vibration movement of protein structure of xanthine oxidoreductase that potentially produces active oxygen species. Outline of the significant results as follows : 1) We have assigned the atom ligands and their geometries of the molybdenum atom of the MoCo center in the catalytic center of xanthine oxidoreductase by X-ray crystallographic analysis. In contrast to the previous reports, comparison between the electron densities of the sulfo and desulfo forms indicated that the oxygen atom locates at the apical position while the sulfo atom locates at the equatorial position. These results account for the hydride transfer mechanism during hydroxylation reaction proposed in the structure of reaction intermediate of the enzyme with slow substrate of FYX-051. We also proposed the role of amino acid residues located in the active site cavities in the hydroxylation of purine substrates by site directed mutagenesis experiments. 2) We have revealed the overall mechanism of the conversion from the dehydrogenase to the oxidase revealed by the three-dimensional structures analysis, and have published the invited review papers. 3) In order to account for the different effects of inhibitors on the mammalian and bacterial enzymes observed in the anti-gout drugs, we have determined the molecular dynamics by computer simulation analysis. The results suggested that the dynamics of the hydrophobic peptide exist in the cavity differ significantly between bacterial and mammalian enzymes. 4) We have constructed transgenic mouse that produces large amount of superoxide compare to the normal enzyme. Analyses of phenotype are still under way. We have analyzed the effects of inhibitors of xanthine oxidoreductase on the ALS model mouse, it was found that some of the inhibitors are quite effective for therapeutic usage.