Studies on site directed mutant pig citrate synthases.
Studies on site directed mutant pig citrate synthases.
复制标题
定点突变猪柠檬酸合酶的研究。
DOI：
10.1016/0006-291x(89)91831-7
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发表时间：
1989
影响因子：
3.1
通讯作者：
Srere,PA
中科院分区：
文献类型：
--
作者：
Evans,CT;Owens,D;Casazza,JP;Srere,PA
将编码猪柠檬酸合酶（PCS）非突变型和突变型的DNA亚克隆到表达系统中，以测定它们在E.大肠杆菌gltA细胞中存在细菌柠檬酸合酶缺陷。表达非突变体PCS DNA的GltA细胞在限定的最小乙酸盐培养基上生长，并产生恒定水平的PCS（0.43 U/mg蛋白质）。相反，当用编码His 274或Asp 375中PCS突变的DNA转化gltA细胞时，细胞不在最低乙酸盐培养基上生长。突变体PCS蛋白在大肠杆菌中的存在。通过蛋白质印迹和使用猪心柠檬酸合酶特异性抗体的免疫分离分析证实了对大肠杆菌的感染。在总细胞裂解物中，突变体PCS酶的活性比非突变体酶低两个数量级。这些数据表明，活性位点氨基酸，His 274和Asp 375，是必不可少的柠檬酸合成酶的催化活性。
The DNAs encoding the non-mutant and mutant forms of pig citrate synthase (PCS) were subcloned into an expression system to determine their synthesis and stability in E. coli gltA cells that are defective in bacterial citrate synthase. GltA cells that expressed the non-mutant PCS DNA grew on defined minimal acetate media and produced a constant level of PCS (0.43 U/mg protein). In contrast, when the gltA cells were transformed with the DNA encoding PCS mutations in His274 or Asp375 the cells did not grow on minimal acetate media. The presence of the mutant PCS proteins in E. coli was confirmed by protein blot and immunoisolation analyses using an antibody specific for porcine heart citrate synthase. The activities of the mutant PCS enzymes were two orders of magnitude less than the non-mutant enzyme in the total cell lysates. The data indicate that the active site amino acids, His274 and Asp375, are essential for the catalysis activity of citrate synthase.