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

钼酶精细结构的测定以及羟基化和蛋白质振动的机制。

基本信息

批准号：
16205021
负责人：
NISHINO Takeshi
金额：
$ 30.87万
依托单位：
Nippon Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2007
项目状态：
已结题

项目摘要

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.

我们一直致力于黄嘌呤氧化还原酶蛋白质结构的构象变化和振动运动，从而可能产生活性氧。重要结果概述如下： 1）通过X射线晶体学分析，我们确定了黄嘌呤氧化还原酶催化中心MoCo中心钼原子的原子配体及其几何形状。与之前的报道相反，磺基和脱磺基形式的电子密度之间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慢底物酶的反应中间体结构中提出的羟基化反应期间的氢化物转移机制。我们还通过定点诱变实验提出了位于活性位点空腔中的氨基酸残基在嘌呤底物羟基化中的作用。 2）我们通过三维结构分析揭示了脱氢酶向氧化酶转化的整体机制，并发表了特邀综述论文。 3）为了解释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，细菌和哺乳动物酶之间存在的疏水性肽的动力学存在显着差异。 4）我们构建了与正常酶相比产生大量超氧化物的转基因小鼠。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，发现某些抑制剂对于治疗用途相当有效。