大肠杆菌精氨酰－tRNA合成酶的Lys378和381的点突变研究

用点突变的方法将大肠杆菌精氨酰—ｔＲＮＡ合成酶（ＡｒｇＲＳ）的基因ａｒｇｓ上相应于Ｌｙｓ３７８和Ｌｙｓ３８１的密码ＡＡＡ分别变为两氨酸的密码ＧＣＡ和精氨酸的密码子ＣＧＴ，得到了４个ａｒｇｓ的突变体ａｒｇｓ３７８ＫＡ，ａｒｇｓ３７８ＫＲ，ａｒｇｓ３８１ＫＡ和ａｒｇｓ３８１ＫＲ，将它们分别连接到ｐＵＣ１８上，转入大肠杆菌ＴＧ１，在ＴＧ１转化子中，ＡｒｇＲＳ及其变种的表达量约为ＴＧ１中的１２０倍以上。结果表明Ｌｙｓ３７８为Ａｒｇ和Ａｌａ取代分别使活力下降０％和１０％；Ｌｙｓ３８１变为Ａｌａ和Ａｒｇ后，活力分别下降３３％和１０％左右。Ｌｙｓ３７８不为酶活力必需。Ｌｙｓ３８１部位的正电荷对酶活力是重要的。

The Role of Lys378 and Lys381 on the Enzyme Activity of E. coli Arginyl-tRNA Synthetase

By site-directed mutagenesis the codon of Lys 378 and Lys 381,AAA in E. coli gene of arginyl-tRNA synthetase(args) was changed to ICA coding for Ala and CGT for Arg, respectively and four mutants of args 378 Km,augs 378KR, args 381KA and args 381KR were obtained.The mutants were ligated in the gap between BamHI/HindIII of pUC18 and transformed into E.coli TG1.The expressions of arginyl-tREA synthetase(ArgRS) and its mutants in TG1 transformats were 120 times higher than that in TG1.The result has shown that the activity of this enzyme was not decreased or decreased only 10% by the Teplacement of Arg and Ala for Lys 378, respectively.The activity of AceRS decreased 33% and 10% as Lys 381 was changed into Ala or Arg,respectively.Thus,acs 378 is not necessary for the aetivity of this enzyme while the positive charge in 381 residue is important for the activity of ArgRS.