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

硒代半胱氨酸残基作为一个完整的片段出现在含硒酶的活性中心，如甘氨酸还原酶、甲酸脱氢酶和谷胱甘肽过氧化物酶。硒半胱氨酸残基的催化作用是由于硒半胱氨酸残基的硒醇基团具有高反应活性：硒醇的氧化还原电位远低于相应的硫。因此，人们关注于合成含有自去半胱氨酸的多肽和蛋白质，这些多肽和蛋白质可能具有硫对应物所不具有的独特性质。本文合成了新型含硒半胱氨酸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）是一类低分子量、富含半胱氨酸的蛋白质，可与锌、镉、铜等金属离子结合。Neuros ora Cu-MT仅由25个氨基酸组成，并将6个Cu离子与7个半胱氨酸残基结合…更多信息：迄今为止分离的最小MT。我们合成了MT肽的硒类似物（硒金属硫蛋白（selometallothionein, SeMT）），其中所有的半胱氨酸残基都被相应的硒取代，以研究SeMT- cu配合物中硒酸铜簇的结构。与天然的Cu- mt配合物相反，3个Cu离子以单个Cu(II)-硒酸盐簇的形式配位。为了进一步了解复合物的结构，我们尝试了EXAFS分析。MT和SEMT的Cu配合物的XANES振荡显示出显著的相似性。这表明CU（II）硫代酸盐簇在CuMT中的结构与CU(II) -硒酸盐簇在硒对应物中的结构相似。基因编码。为神经孢子虫合成了MT并与β -gal基因融合。mt - β -gal融合基因在大肠杆菌中表达，产生融合蛋白。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融合基因氨基末端附近的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突变基因。该方法基于在延伸反应中使用变性双链DNA作为模板和硫代修饰核苷酸。仅在厌氧培养时检测到β - gal活性，并将硒掺入融合蛋白中。这些结果表明，在厌氧条件下诱导形成的TGA抑制因子调节了SeCys进入蛋白质，这些因子不仅将SeCys引入到固有的含有TGA密码子编码的SeCys的蛋白质中，而且通过将TGA人工引入到其他不含SeCys的蛋白质中。硒谷胱甘肽是谷胱甘肽的硒类似物，用应用生物系统多肽合成器430A合成。合成的化合物为氧化态。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