Selenium Biochemistry

硒磷酸合成酶（SPS）将ATP和硒转化为硒磷酸，硒磷酸是哺乳动物和几种细菌合成硒酶的硒供体。马特·沃尔夫博士发现，纯化的大肠杆菌酶含有一种以前未检测到的结合发色团，可以吸收紫外线。这种发色团已被检测为一种不太稳定的衍生物，与蛋白质水解裂解产生的肽结合。对该发色团的身份及其在催化反应机制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，成功地结晶出了一种突变的SPS。这是特别令人印象深刻的，因为许多研究人员早期结晶SPS的所有尝试都失败了。产生了一种以硒代蛋氨酸取代蛋氨酸残基的SPS蛋白形式，该衍生物的晶体将进行x射线分析。硒代蛋氨酸可作为计算晶体中其他残基距离的参考点。通过这些方法可以得到一些关于SPS活性位点的位置和氨基酸组成的信息。

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.