Study of Selenium-Dependent Xanthine Dehydrogenase and Purine Hydroxylase

尽管最近在硒蛋白中结合硒的研究取得了很大进展，但这项工作的重点主要放在以硒半胱氨酸形式携带硒的酶上。少数几种酶的特征是，硒存在并需要活性，但在mRNA翻译过程中不插入，因此不以硒半胱氨酸的形式存在。两种这样的酶是来自梭状芽孢杆菌的烟酸羟基酶(NAH)和黄嘌呤脱氢酶(XDH)。NaH中存在的硒是不稳定的，可以用促进剂，如十二烷基硫酸钠处理来去除。为了更好地了解硒的性质和硒的掺入情况，从紫质溶血梭菌中分离纯化了XDH。经SDS-PAGE分析，XdH由3个亚基组成，并用75-Se标记。这种硒是不稳定的，推测不是以硒半胱氨酸的形式存在。在分离该酶的过程中，还分离到了另一种含硒羟基酶，命名为嘌呤羟基酶(PH)。PH由四个亚基组成(变性凝胶电泳法)，以嘌呤和次黄嘌呤为底物。PH中的硒也是不稳定的，因此代表了另一种需要表征和用于这些独特酶的比较分析的非硒半胱氨酸需硒酶。既然已经确定了从紫色葡萄球菌中分离XDH和PH的较好的纯化方案，那么这些酶将进行更详细的研究。这些研究将集中于鉴定辅因子和存在的金属，以及利用电子顺磁共振(EPR)和X射线吸收光谱(XAS)等方法研究酶反应和电子转移的活性中心。调查的重点将围绕确定活性部位中存在的硒的部分及其与其他辅因子(如钼辅因子)的关系。一旦对这些酶中硒的性质有了更多的了解，细胞将硒结合到这个活性部位的机制将被研究。这项工作可能会发现一种新的递送蛋白(S)或硒供体分子，这是XDH和PH以及可能的其他钼羟基酶所必需的，也可能有助于揭示硒在硒半胱氨酸生物合成中的递送。

Although significant knowledge has recently been gained on the incorporation of selenium into selenoproteins, most of the emphasis of this work has been placed on enzymes that carry selenium in the form of selenocysteine. A handful of enzymes have been characterized in which selenium is present and required for activity but is not inserted during translation of the mRNA and therefore is not present as selenocysteine. Two such enzymes are nicotinic acid hydroxylase (NAH) and xanthine dehydrogenase (XDH) from Clostridium. Selenium present in NAH is somewhat labile and can be removed by treatment with chaotropic agents, such as sodium dodecyl sulfate. In order to better understand the nature of selenium and the incorporation of selenium, XDH from Clostridium purinolyticum was purified to homogeneity. XDH was found to consist of three subunits (based on SDS-PAGE) and to be labeled with selenium (75-Se). This selenium was labile and presumably is not in the form of selenocysteine. During the isolation of this enzyme, another selenium-containing hydroxylase was isolated and termed purine hydroxylase (PH). PH consisted of four subunits (based on denaturing gel electrophoresis) and utilized purine and hypoxanthine as substrates. Selenium in PH was also labile, and thus represents yet another non-selenocysteine selenium-requiring enzyme to be characterized and to be used in a comparative analysis of these unique enzymes. Now that a good purification scheme has been determined for the isolation of both XDH and PH from C. purinolyticum, these enzymes will be investigated in more detail. These investigations will focus on the identification of cofactors and metals present, as well as study of the active sites for enzyme reaction and electron transfer using methods such as electron paramagnetic resonance (EPR) and x-ray absorption spectroscopy (XAS). The focus of the investigation will revolve around determining the moiety of selenium present in the active site and its relationship to other cofactors (e.g. molybdenum cofactor). Once more knowledge is gained into the properties of selenium in these enzymes, the mechanism by which the cell incorporates selenium into this active site will be investigated. This work may uncover a new delivery protein(s) or selenium donor molecule required for XDH and PH and possibly other molybdenum hydroxylases, and perhaps shed light on the delivery of selenium in the biosynthesis of selenocysteine.