Evans C T, Owens D, Casazza J P, Srere P A
Department of Veterans Affairs, Dallas, Texas.
Biochem Biophys Res Commun. 1989 Nov 15;164(3):1437-45. doi: 10.1016/0006-291x(89)91831-7.
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.
将编码猪柠檬酸合酶（PCS）非突变型和突变型的DNA亚克隆到一个表达系统中，以确定它们在细菌柠檬酸合酶有缺陷的大肠杆菌gltA-细胞中的合成和稳定性。表达非突变型PCS DNA的gltA-细胞在限定的基本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）。相比之下，当用编码His274或Asp375中PCS突变的DNA转化gltA-细胞时，这些细胞在基本乙酸盐培养基上无法生长。使用针对猪心脏柠檬酸合酶的特异性抗体，通过蛋白质印迹和免疫分离分析证实了大肠杆菌中突变型PCS蛋白的存在。在总细胞裂解物中，突变型PCS酶的活性比非突变型酶低两个数量级。数据表明，活性位点氨基酸His274和Asp375对柠檬酸合酶的催化活性至关重要。
