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

硒代半胱氨酸残基作为含硒酶如甘氨酸还原酶、甲酸脱氢酶和谷胱甘肽过氧化物酶的活性中心中的完整部分存在。硒代半胱氨酸残基的催化作用归因于硒代半胱氨酸残基的硒醇基团的高反应性：硒醇具有比硫对应物低得多的氧化还原电位。因此，已经注意合成含硒半胱氨酸的多肽和蛋白质，其可能具有硫对应物所不具有的独特性质。本研究旨在合成新的含硒半胱氨酸的多肽，硒金属硫蛋白和硒谷氨酸，并对其结构和功能进行研究.金属硫蛋白（Metallothionein，MTs）是一类富含半胱氨酸的低分子量蛋白质，能与锌、镉、铜等金属离子结合。Neurosora Cu-MT由25个氨基酸组成，6个Cu离子与7个半胱氨酰残基结合， 关于我们 UEs：迄今为止隔离的最小MT。我们合成了MT肽的硒类似物硒硫蛋白（SeMT），其中所有的半胱氨酸残基都被硒对应物取代，以研究SeMT-Cu络合物中Cu-硒簇的结构。与天然Cu-MT络合物相反，3个Cu离子以单个Cu（II）-硒簇的形式配位。为了进一步了解复杂的结构，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CuMT中Cu（II）硫醇簇的结构与硒对应物中Cu（II）-硒簇的结构相似.基因编码。合成了链孢霉MT，并与β-gal基因融合。将MT-beta-gal融合基因在E.大肠杆菌以产生融合蛋白。用融合蛋白制备的MT的光谱特征与相应的天然MT的光谱特征相似。将编码MT-β-gal融合基因中靠近氨基末端的第一个半胱氨酸残基的TGT替换为TGA。该突变基因是通过一种新开发的简单方法构建的。该方法是基于在延伸反应中使用变性的双链DNA作为模板和硫代磷酸酯修饰的核苷酸。当仅厌氧培养时，在转化的中检测到β-Gal活性，并且硒被掺入融合蛋白中。这些结果表明，在厌氧条件下诱导形成的TGA抑制因子调节SeCys掺入蛋白质，这些抑制因子不仅将SeCys引入由TGA密码子编码的固有SeCys蛋白质中，而且通过人工将TGA引入基因中将SeCys引入其他不含SeCys的蛋白质中.用Applied Biosystems肽合成仪430 A合成硒谷氨酰胺，谷胱甘肽的硒类似物。合成的化合物为氧化形式。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