MICROBIAL AND ENZYMATIC SYNTHESIS OF SELENIUM-CONTAINING POLYPEPTIDES AND THEIR ACTION

在甘氨酸还原酶、甲酸脱氢酶和谷胱甘肽过氧化物酶等含硒酶的活性中心，硒半胱氨酸残基作为一个完整的部分存在。硒半胱氨酸残基的催化作用归因于硒半胱氨酸残基中硒醇基的高反应性：硒醇的氧化还原电位远低于硫的氧化还原电势。因此，含硒半胱氨酸的多肽和蛋白质的合成受到了人们的关注，这些多肽和蛋白质可能具有硫化合物所没有的独特性质。本研究合成了新型含硒半胱氨酸多肽--硒金属硫蛋白和硒谷胱甘肽，并对其结构和功能进行了研究。金属硫蛋白(MTS)是一类低分子、富含半胱氨酸的蛋白质，与锌、镉、铜等大量金属离子结合。神经细胞铜-MT仅由25个氨基酸组成，6个铜离子与7个半胱氨基残基…结合更多的UE：迄今为止最小的MT。我们合成了金属硫蛋白(SeMT)的硒类似物，其中所有的半胱氨酸残基都被硒取代，以研究SEMT-铜络合物中的铜-硒酸簇的结构。与天然的铜-金属络合物不同，3个铜离子以单一的铜(II)-硒酸络合物的形式配位。为了进一步了解该复合体的结构，我们尝试了EXAFS分析。MT和SEMT的铜配合物的XANES振荡显示出显著的相似性。这表明CuMT中的铜(II)硫酸盐簇合物的结构与硒中的铜(II)-硒酸根簇合物的结构相似。基因编码。对于神经孢子虫，合成了MT，并将其与β-半乳糖基因融合。将MT-β-Gal融合基因在大肠杆菌中表达，获得融合蛋白。用融合蛋白制备的MT的光谱特征与相应的天然MT相似。编码MT-β-Gal融合基因氨基末端第一个半胱氨酸残基的TGT被TGA取代。用一种新开发的简单方法构建了突变基因。该方法是基于以变性双链DNA为模板，在延伸反应中使用硫代磷酸修饰的核苷酸。在厌氧培养条件下，转化菌株中检测到β-半乳糖酶活性，融合蛋白中掺入了硒。这些结果表明，在厌氧条件下诱导形成的TGA抑制因子调节SeCys在蛋白质中的掺入，这些因子不仅将SeCys引入到由TGA密码子编码的固有含SeCys的蛋白质中，而且通过人工将TGA引入到基因中，将SeCys引入到其他不含SeCys的蛋白质中。应用生物系统多肽合成仪430A合成了谷胱甘肽的硒类似物--硒谷胱甘肽。合成的化合物为氧化形式。较少

A selenocysteine residue occurs as an integral Moiety in the active center of the selenium-containing enzymes such as glycine reductase, formate dehydrogenase and glutathione peroxidase. The catalytic role of the seleno- cysteine residue is attributed to high reactivity of the selenol group of the selenocysteine residues: selenols have much lower redox potential than the sulfur counterparts. Thus, attention has been paid to synthesis of sele- nocysteine-containing polypeptides and proteins which may have unique properties that the sulfur counterparts do not have. The present study was performed to synthesize the novel selenocysteine-containing peptides, seleno- metallothionein and selenoglutathione, and to elucidate their structures and function.1. Metallothioneins (MTs) are a class of low-molecular weight and cysteine rich proteins, which bind with high amount of metal ions such as Zn, Cd and Cu. Neuros ora Cu-MT consists of only 25 amino acids and binds 6 Cu ions to 7 cysteinyl resid … More ues: the smallest MT thus far isolated. We synthesized selenium analogue of the MT peptide (selenometallothionein (SeMT)), in which all the cysteine residues are replaced by selenium counterparts, to study the structure of the Cu-selenoate cluster in a SeMT-Cu complex. In contrast to the native Cu-MT complex, 3 Cu ions are coordinated in the form of a single Cu(II)-selenoate cluster. To obtain further insight into the structure of the complex, we have attempted EXAFS analysis. The XANES oscillations of the Cu complexes of MT and SEMT revealed a remarkable similarity. This indicates that the structure of CU(II) thiolate cluster in the CuMT is similar to that of Cu(II)-selenoate cluster in the selenium counterpart.2. The gene coding. for the Neurospora MT was synthesized and fused with the gene of beta-gal. The MT-beta-gal fused gene was expressed in E. coli to produce a fused protein. The spectral features of the MT prepared with the fused protein were similar to those of the corresponding native MTs. TGT encoding the first cysteine residue near the amino terminal in the MT-beta-gal fused gene was replaced by TGA. The mutant gene was constructed by a simple method newly developed. The method is based on the use of denatured double-stranded DNA as a template and phosphorothioate-modified nucleotide in the elongation reaction. beta-Gal activity was detected in the transformed when cultivated anaerobic only, and selenium was incorporated into the fused protein. These results suggest that the TGA suppression factors formed inducibly under anaerobic conditions regulate SeCys incorporation into proteins, and that these factors introduce SeCys not only into inherent SeCys-containing proteins whose SeCys is encoded by the TGA codon, but also into other proteins that have no SeCys by introduction of TGA into the gene artificially.3. Selenoglutathione, a selenium analogue of glutathione was synthesized with an Applied Biosystems peptide synthesizer 430A. The compound synthesized was oxidized form. Less