Incorporation of Tellurocysteine into Glutathione Transferase Generates High Glutathione Peroxidase Efficiency
Incorporation of Tellurocysteine into Glutathione Transferase Generates High Glutathione Peroxidase Efficiency
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将碲半胱氨酸掺入谷胱甘肽转移酶可产生高谷胱甘肽过氧化物酶效率
DOI：
10.1002/anie.200805365
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发表时间：
2009-01-01
影响因子：
16.6
通讯作者：
Shen, Jiacong
中科院分区：
文献类型：
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作者：
Liu, Xiaoman;Silks, Lottis A.;Shen, Jiacong
氧化应激直接或间接地与一系列人类疾病的病理有关：细胞内氧化物质浓度的增加导致膜、蛋白质、DNA的氧化，并最终导致细胞死亡因此，开发用于医疗的高效抗氧化剂变得越来越重要。谷胱甘肽过氧化物酶（gpx）1.9)在哺乳动物中首次被发现，是参与清除活性氧的关键酶。它们有效的抗氧化活性取决于在催化位点存在的稀有氨基酸残基硒代半胱氨酸（SeCys）。这种残留物在催化循环中依次被氧化，然后被还原由于天然GPx的固有缺点限制了其应用，因此对GPx的功能进行了大量的模拟研究。例如，低分子GPx模拟物2-苯基- 1,2 -苯并二苯并异硒唑-3 (2H)- 1 （ebselen）已被临床评估用于治疗中风，[3]和一系列低分子含硒/碲化合物和化学修饰的含硒生物大分子已被报道为GPx模拟物。[3,4]然而，由于缺乏底物谷胱甘肽（GSH）的结合位点，当GSH作为还原底物时，模拟物的活性有限。利用单克隆抗体和生物印迹技术将GSH结合位点成功导入GPx模型，得到的[5]和含硒蛋白具有较高的GPx活性。在谷胱甘肽s -转移酶（GST, EC 2.5）中，谷胱甘肽结合域折叠之间惊人的整体结构相似性。1.18)和GPx，以及它们催化中心取向的相似性，导致选择GST作为诱导GPx功能的优秀支架Lucilia cuprina的GST必需活性位点Ser9 （LuGST1-1）被一个SeCys残基取代，得到GPx模拟物硒-LuGST1-1，它以GSH为还原底物表现出较高的活性与关于硒在生物系统中的结合的大量知识相比，令人惊讶的是，对碲的发生所知甚少。telluro半胱氨酸（TeCys）[8]具有本质上较低的氧化还原电位（À850 mV vs Ag/AgCl）比SeCys （GPx的催化中心；À640 mV vs Ag/AgCl）因此，含有TeCys残基的蛋白质可以参与独特的生物学基础氧化还原反应。碲蛋氨酸已被纳入蛋白质中进行结构分析将TeCys化学引入枯草菌素中形成半合成的tellurosubilisin是唯一被报道的TeCys掺入的例子然而，化学修饰有一个明显的缺点：只能修饰活性丝氨酸残基（如枯草菌素中的Ser221）；其他丝氨酸残基是不可接近的。因此，开发一种将TeCys结合到蛋白质中以获得碲化酶的通用策略提出了一个巨大的挑战。通过使用营养缺陷表达系统，我们成功地将TeCys残基整合到蛋白中（图1）。合理设计的telluro酶将现有的GSH结合位点与催化的TeCys残基结合在一起，显示出与天然GPx相媲美的显著GPx活性。
Oxidative stress is implicated, either directly or indirectly, in the pathology of a range of human diseases: An increase in intracellular concentrations of oxidizing species leads to the oxidation of membranes, proteins, DNA, and ultimately to cell death.[1] As a consequence, the development of efficient antioxidants for medical use has become increasingly important. Glutathione peroxidases (GPxs, EC 1.11. 1.9) were first discovered in mammals as key enzymes involved in the scavenging of reactive oxygen species. Their efficient antioxidant activity depends on the presence of the rare amino acid residue selenocysteine (SeCys) at the catalytic site. This residue is successively oxidized and then reduced during catalytic cycles.[2] As intrinsic disadvantages of natural GPxs have limited their application, enormous efforts have been made to simulate the functions of GPx. For example, the lowmolecular-weight GPx mimic 2-phenyl-1, 2-benzoisoselenazol-3 (2H)-one (ebselen) has already been evaluated clinically for the treatment of stroke,[3] and a series of low-molecularweight selenium/tellurium-containing compounds and chemically modified selenium-containing biomacromolecules have been reported as GPx mimics.[3, 4] However, because of the absence of the binding site for substrate glutathione (GSH), the mimics displayed limited activity when GSH was used as the reducing substrate. The GSH binding site was introduced successfully into GPx models by using monoclonal antibody and bioimprinting techniques,[5] and the resulting seleniumcontaining proteins exhibited high GPx activity. The striking overall structural similarity between the glutathione-bindingdomain folds in glutathione S-transferase (GST, EC 2.5. 1.18) and GPx, and the similarity in the orientation of their catalytic center, led to the selection of GST as an excellent scaffold for inducing GPx function.[6] The essential active-site residue Ser9 of GST from Lucilia cuprina (LuGST1-1) was replaced with an SeCys residue to give the GPx mimic seleno-LuGST1-1, which exhibits high activity with GSH as the reducing substrate.[7] In contrast to the vast body of knowledge with regard to selenium incorporation in biological systems, surprisingly little is known about the occurrence of tellurium.Tellurocysteine (TeCys)[8] has an intrinsically lower redox potential (À850 mV versus Ag/AgCl) than that of SeCys (the catalytic center of GPx; À640 mV versus Ag/AgCl).[9] As a result, proteins containing TeCys residues can participate in unique and biologically fundamental redox reactions. Telluromethionine has been incorporated into proteins for structure analysis.[10] The chemical introduction of TeCys into subtilisin to form semisynthetic tellurosubtilisin is the only example of TeCys incorporation to have been reported.[4] However, chemical modification has an obvious disadvantage: Only the active serine residue (eg Ser221 in subtilisin) can be modified; other serine residues are not accessible. Thus, the development of a general strategy for the incorporation of TeCys into proteins to give telluroenzymes poses a great challenge. By using an auxotrophic expression system, we have successfully incorporated TeCys residue into a protein (Figure1). The rationally designed telluroenzyme, which combines an existing GSH binding site with a catalytic TeCys residue, exhibits a remarkable GPx activity that rivals that of native GPxs.