大肠杆菌亮氨酰－tRNA合成酶LeuRS67R的纯化及其动力学研究  

^The Purification and Studies of Kinetics of E. coli Leucyl-tRNA Synthetase Mutant (LeuRS67R)

作　　者：李彤[1] 夏宪[1] 王恩多[1] 王应睐 

机构地区：[1]中国科学院上海生物化学研究所分子生物学国家重点实验室

出　　处：《生物化学与生物物理学报》1995年第3期279-285,共7页

基　　金：国家自然科学基金

摘　　要：本实验室已得到的亮氨酰－ｔＲＮＡ合成酶（ＬｅｕＲＳ）基因，与文献相比，６７位氨基酸残基由Ｈｉｓ变为Ａｒｇ，此酶被定名为ＬｅｕＲＳ６７Ｒ。我们从该基因与ｐＵＣ１９重组质粒的大肠杆菌ＴＧ１转化子ＴＧ１－９１中得到ＬｅｕＲＳ的高表达，粗抽液中ＬｅｕＲＳ的表达量在转化子中比在宿主菌ＴＧ１中高２０倍以上。用三步拉层析得到电泳一条带的酶，其比活为１７８９单位／毫克。测定其动力学常数，氨酰化活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０２７ｍｍｏｌ／Ｌ、０．４７ｍｍｏｌ／Ｌ，Ｋｃａｔ值分别为３．５～５．１ｓ－１。ＡＴＰ－ＰＰｉ交换活力对Ｌｅｕ、ＡＴＰ的Ｋｍ值分别为０．０３ｍｍｏｌ／Ｌ、１．０ｍｍｏｌ／Ｌ，Ｌｃａｔ值分别为１４０～１５５ｓ－１。此结果与从野生型大肠杆菌Ｋ－１２中提纯的ＬｅｕＲＳ的动力学常数差别很小，６７位氨基酸残基在与活性中心无直接关系的域可能是大肠杆菌的种间差异。The gene coding for leuoyl-tRNA synthetase (LeuRS) has been cloned from E. coli K-12. There is a substitute of G for A at the position of base 200 in the gene compared with the gene from E. ooU K-12 1100. Because His67 of this enzyme is changed to Arg. the enzyme was designated LeuRS67R. We have got over expresaion of this enzyme from E. coli TG1 transformant strain which harbors the recombination plasmid pUC19 with the gene. In the crude extract of the transformant strain LeuRS67R was overproduced 20 times more than that of the wild-type TG1 strain. The purified enzyme showing one band on SDS-PAGE has been obtained by three steps of column chromatography. The specific activity of the preparation was 1786 units/mg. For the andnoacylation reaction the K values at pH7.4, 37℃, for Leu and ATP were determined to be 0.027 mM and 0.47 mM, respectively, kcat was 3 .5￣5 .1s-1. The Km values for ATP-PPi exehange reaction were found to be 0 .03 mM and 0.75mM for leucine and ATP, respectively, kcat was 140￣155s-1. The result has shown there is a little difference in the kinetic constants between LeuRS67R and LeuRS previously obtained from E.coli K-12.This mutant at 67 amino acid residue may not be involved in the active Bite.

关 键 词：大肠杆菌 动力学常数 亮氨酰-tRNA 合成酶 

分 类 号：Q939.110.6[生物学—微生物学]