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Creation and design of novel enzymes by analysis and modification of nucleotide sequence in DNA and its application

通过DNA核苷酸序列的分析和修饰来创建和设计新型酶及其应用

基本信息

批准号：
61480057
负责人：
KIMURA Akira
金额：
$ 3.33万
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 (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

项目摘要

Glutathione synthetase (GSH-II) of Eschrichia coli B catalyzes the synthesis of glutathione from <gamma>-glutamylcysteine and glycine in the Presence of adenosine 5'-triphosphate (ATP).To analyze the relation between function and structure of the enzyme,we first searched for proteins with a homologous amino acid sequence with that of GSH-II and found that an enzyme,dihydroxyfolate reductase(FDR)has a region approximately 70% homologous in amino acid sequence.The enzymatic analyses using inhibitors and/or substrates of the two enzymes (GSH-II and FDR) indicated that the homologous region represented the catalytic domain on the two enzymes.Secondly,to know the contribution of cysteine residues on activity and/or stability of the GSH-II,the 4 codons for cysteine in the GSH-II subunit were severally changed to the codons for alanine by site-directed mutagenesis.Although either replacement did not affect the activity,the replacements of more than 2 cysteine residues resulted in the significant decrease in the GSH-II activity. The 4 cysteine residues in the GSH-II subunit were,therefore,thought to play an important role in the full expression of the GSH-II activity.<gamma>-Glutamylcysteine synthetase(GSH-I)catalyzes the first step of glutathione synthesis and produces <gamma>-glutamylcysteine from glutamate and cysteine in the presence of ATP.The gene for the GSH-I has an unusual initiation codon TTG.The codon (TTG)was replaced with general initition codon ATG by site-directed mutagenesis.As a result, the transcriptional efficiency of the gene was increased more than 50%.

大肠杆菌B的谷胱甘肽合成酶（GSH-II）在<gamma>5 '-三磷酸腺苷（ATP）存在下催化γ-谷氨酰半胱氨酸和甘氨酸合成谷胱甘肽。为了分析该酶的功能与结构的关系，我们首先寻找与GSH-II具有同源氨基酸序列的蛋白质，二羟叶酸还原酶（dihydroxyfolate reductase，FDR）在氨基酸序列上有约70%的同源性，用这两种酶的抑制剂和/或底物进行酶促分析第二，为了了解半胱氨酸残基对GSH-Ⅱ活性和/或稳定性的贡献，通过定点突变将GSH-Ⅱ亚基中的4个半胱氨酸密码子分别改变为丙氨酸密码子，虽然这两种改变都不影响活性，超过2个半胱氨酸残基的替换导致GSH-II活性的显著降低。因此，认为GSH-II亚基中的4个半胱氨酸残基在GSH-II活性的充分表达中起重要作用。<gamma>- 谷氨酰半胱氨酸合成酶（GSH-I）催化谷胱甘肽合成的第一步，<gamma>在ATP存在下由谷氨酸和半胱氨酸合成-谷氨酰半胱氨酸。GSH-I基因具有一个特殊的起始密码子TTG。通过定点突变，将TTG密码子替换为通用起始密码子ATG，使该基因的转录效率提高50%以上。