大肠杆菌精氨酰－tRNA合成酶的Lys306为酶活力所必需

将大肠杆菌精氨酰ｔＲＮＡ合成酶（ＡｒｇＲＳ）上Ｌｙｓ３０６用基因点突变的方法分别变为Ａｌａ和Ａｒｇ的密码子；得到变种基因ａｒｇｓ３０６ＫＡ和ａｒｇｓ３０６ＫＲ。变种基因重组在ｐＵＣ１８上，转化到大肠杆菌ＴＧ１中，转化子中ＡｒｇＲＳ及其变种ＡｒｇＲＳ３０６ＫＡ和ＡｒｇＲＳ３０６ＫＲ所表达的蛋白量至少为ＴＧ１表达ＡｒｇＲＳ蛋白量的１００倍。细胞粗抽提液中ＡｒｇＲＳ的比活ＴＧ１、转化子ｐＵＣ１８－ａｒｇｓ、ｐＵＣ１８－ａｒｇｓ３０６ＫＡ和ｐＵＣ１８－ａｒｇｓ３０６ＫＲ分别为１．６５、２１０、１．８和３８单位／毫克。结果表明ＡｒｓＲＳ的Ｌｙｓ３０６为Ａｌａ取代使活力完全丧失；若被Ａｒｇ取代，则活力丧失８０％以上。Ｌｙｓ３０６为ＡｒｇＲＳ活力所必需。

Lys 306 of E.coli Arginyl-tRNA Synthetase is Necessary for the Activity of This Enzyme

Lys 306 in E.coli arginyl-tRNA synthetase(ArgRS) was changed to Ala and Arg by site-directed mutagenesis.The mutant genes of ArgRS(args),args306KA and args306KR,were obtained and inserted in the vector pUC18 to transform E.coli TG1.The transformats containing the recombinant plasmids pUC18-args,pUC18-args306KA and pUC18-arg306KR,ArgRS overproduced 100 times than E.coli TG1.In the crude extract of E.coli TG1 and its transformats harbouring the recombinant plasmids,pUC18-args,pUC18-args 306KA and pUC18-argsKR, the specific activities of ArgRS were 1.65,210,1.8 and 38 unit/mg,respectively.The result showed that by the replacement of Lys 306 of ArgRS with Ala and Arg,the activity of ArgRS was lost completely and 80% respectively. Lys306 was necessary for the activity of ArgRS.