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APPLICATION OF GLUTATHIONE SYNTHETASE ON PEPTIDE SYNTHESIS

谷胱甘肽合成酶在肽合成中的应用

基本信息

批准号：
03660138
负责人：
ODA Jun'ichi
金额：
$ 1.34万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

We have been studying the crystal structure of glutathione synthetase (GSHase) -substrates complex. These studies showed that the binding site of ATP is between two anti-parallel beta-sheets and that some residues interact with the bound ATP. However, the binding site of gamma-Glu- Cys is not clear because gamma-Glu-Cys gave poor electron density. We assumed some residues in the putative binding site of gamma-Glu-Cys and investigated their properties on the catalysis. Some mutant GSHases in which the residues around proposed binding site of gamma-Glu-Cys are replaced by site- directed mutagenesis are prepared and investigated in their properties. The analysis of the mutant GSHases shows that gamma-Glu-Cys was binding between Arg86 and Arg210 and that the thiol group of gamma-Glu-Cys was recognized by Thr288.The binding site of ATP was confirmed by the technique of affinity labeling. The crystallography of the labeled GSHase showed that the binding site of the modifier (adenosine-5'-tetraphospho-5'-pyridoxal) is the same as that of ATP.Crystallography of glutathione synthetase showed that a loop from Ile226 to Gly242 is above the binding sites and gave no electron density because of its flexibility. The position of the loop suggested that the loop have some roles on the catalysis. The analysis of the loop indicated that the loop moved over the active site to protect a labile intermediate from hydrolysis, controlled the recognition of glycine, and accelelared the catalysis by aligning the substrates in proper position.These results suggest that the mutation of residues around the binding sites and of the loop give new enzymes which catalyze synthesis of a novel peptide.

我们一直在研究谷胱甘肽合成酶(GSHase) -底物复合物的晶体结构。这些研究表明ATP的结合位点在两个反平行的β -片之间，并且一些残基与结合的ATP相互作用。由于γ - glu -Cys的电子密度较低，因此γ - glu -Cys的结合位点不清楚。我们假设γ - glu - cys的结合位点上有一些残基，并研究了它们的催化性质。制备了一些γ - glu - cys结合位点周围的残基被位点定向诱变所取代的突变型gshase，并对其性质进行了研究。突变体GSHases分析表明，γ - glu - cys在Arg86和Arg210之间结合，并且γ - glu - cys的巯基被Thr288识别。通过亲和标记技术确定了ATP的结合位点。标记GSHase的晶体学表明，修饰物（腺苷-5′-四磷酸-5′-吡哆醛）的结合位点与ATP的结合位点相同。谷胱甘肽合成酶的晶体学表明，在结合位点上方有一个从Ile226到Gly242的环，由于其柔韧性，没有电子密度。环的位置表明该环在催化过程中有一定的作用。对该环的分析表明，该环在活性位点上移动以保护不稳定中间体不被水解，控制对甘氨酸的识别，并通过在适当的位置对齐底物来加速催化。这些结果表明，结合位点周围的残基和环的突变产生了催化合成新肽的新酶。