Selenium Biochemistry

硒磷酸合成酶（SPS）将ATP和硒转化为硒磷酸盐，硒磷酸盐是哺乳动物和几种细菌中硒酶合成的硒供体。马特沃尔夫博士发现，纯化的E。大肠杆菌酶含有一个以前未检测到的结合发色团，吸收紫外线。该发色团已被检测为与蛋白质的蛋白水解切割产生的肽之一结合的稍微不稳定的衍生物。确定这种发色团的身份及其在催化反应机理中可能发挥的作用的研究正在进行中。拉特·瓦塔纳萨克在沃尔夫博士的指导下，成功地结晶出了SPS的突变形式。这是特别令人印象深刻的，因为所有早期的尝试，许多研究人员结晶SPS失败。产生了其中硒代甲硫氨酸取代甲硫氨酸残基的SPS蛋白的形式，并且将对该衍生物的晶体进行X射线分析。硒代甲硫氨酸用作计算晶体中其他残基距离的参考点。通过这些方法可以获得SPS活性位点的位置和氨基酸组成等信息。 在SPS活性的通常体外测定中，添加硒化物（mM高毒性水平）作为硒底物。作为替代方案，已表明某些硒结合蛋白可用于在体外将硒的基本形式递送至SPS。基于拉库尔西埃？甘油醛-3-磷酸脱氢酶（GADPH）是一种糖酵解酶，是大肠杆菌中的一种。大肠杆菌蛋白结合硒在体内，实验设计使用GADPH作为一个潜在的硒在体外传递蛋白。利用从人红细胞中纯化的同源四聚体酶，Yuki Ogasawara博士表明GADPH可以在体外结合硒，并作为有效的硒递送蛋白。孵育与硒二葡糖苷酸（GSSeSG）转化的GADPH的衍生物含有1当量的结合硒每个单体，大概在反应性低pKa半胱氨酸存在于每个亚基。这种硒在体外测定中与ATP一起转移到SPS，并转化为硒磷酸盐。从甲烷球菌中克隆了硒结合蛋白基因，并在大肠杆菌中表达。William Self博士的研究。重组蛋白由8.8 kDa的亚基组成，并且是非常疏水的。Kemberly Patteson博士进行的物理化学研究解释了分离蛋白质保留结合硒的能力。硒结合需要位于每个亚基中的单个半胱氨酸残基。该半胱氨酸残基完全埋藏在氧化和还原形式的蛋白质中，并且有必要部分解折叠蛋白质以检测其化学反应性。Michelle Galloway博士正在进行的实验表明，她制备的含有共价结合的硒磷酸合成酶的亲和基质具有选择性地与硒结合蛋白反应的潜力。在使用GADPH的初步测试中，该蛋白质结合到亲和基质上，并且可以用含有DTT的KCl洗脱。这种方法可能被证明是有用的检测和分离潜在的硒传递蛋白从各种来源。

Selenophosphate synthetase (SPS) converts ATP and selenium to selenophosphate, the selenium donor for selenoenzyme synthesis in mammals and several bacterial species. Dr. Matt Wolfe discovered that the purified E. coli enzyme contains a previously undetected bound chromophore that absorbs in the UV. This chromophore has been detected as a somewhat unstable derivative bound to one of the peptides produced by proteolytic cleavage of the protein. Studies to determine the identity of this chromophore and its possible role in the mechanism of the catalytic reaction are in progress. Rut Wattanasak, under the direction of Dr. Wolfe, has succeeded in crystallizing a mutant form of SPS. This is particularly impressive because all earlier attempts by many investigators to crystallize SPS had failed. A form of the SPS protein in which selenomethionine was substituted for methionine residues was produced and crystals of this derivative will be subjected to X-ray analysis. The selenomethionine serves as a reference point for calculation of distances to other residues in the crystal. Some needed information concerning location and amino acid composition of the active site of SPS may result from these approaches. In the usual in vitro assay for SPS activity, selenide (mM highly toxic levels) is added as the selenium substrate. As an alternative it was shown that certain selenium-binding proteins could be used to deliver an elementary form of selenium to SPS in vitro. Based on Lacourciere?s identification of glyceraldehyde-3-phosphate dehydrogenase (GADPH), a well-known glycolytic enzyme, as one of the E. coli proteins that bound selenium in vivo, experiments were designed to use GADPH as a potential selenium delivery protein in vitro. Using purified homo-tetrameric enzyme from human erythrocytes, Dr. Yuki Ogasawara showed that GADPH could bind selenium in vitro and serve as an effective selenium delivery protein. Incubation with selenodiglutathione (GSSeSG) converted the GADPH to a derivative containing 1 equivalent of bound selenium per monomer, presumably on the reactive low pKa cysteine present in each subunit. This selenium was transferred to SPS in the in vitro assay with ATP and converted to selenophosphate. The gene encoding a selenium-binding protein previously isolated from Methanococcus vannielii was cloned and expressed in E. coli by Dr. William Self. The recombinant protein consists of 8.8 kDa subunits and is very hydrophobic. Physical chemical studies carried out by Dr. Kemberly Patteson served to explain the ability of the isolated protein to retain bound selenium. A single cysteine residue located in each subunit is required for selenium binding. This cysteine residue is completely buried both in oxidized and reduced forms of the protein and it was necessary to partially unfold the protein to detect its chemical reactivity. Experiments in progress by Dr. Michelle Galloway indicate that an affinity matrix containing covalently bound selenophosphate synthetase that she has prepared has the potential to react selectively with a selenium binding protein. In preliminary tests using GADPH, this protein bound to the affinity matrix and could be eluted with KCl containing DTT. This approach may prove useful for detection and isolation of potential selenium delivery proteins from various sources.