Cavallius J, Merrick W C
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
J Biol Chem. 1998 Oct 30;273(44):28752-8. doi: 10.1074/jbc.273.44.28752.
Site-directed mutants of eEF1A (formerly eEF-1alpha) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345-358). The nucleotide specificity sequence NKMD (residues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE). All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant. Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenylalanine similar to wild type, although with a Km for GTP increased by 1-2 orders of magnitude. The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type. The mutant NKMW eEF1A showed unusual properties. The yeast strain was temperature sensitive for growth, although the purified protein was not. Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type. In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.
使用一种经过改良的高度通用的酵母穿梭载体（Cavallius, J., Popkie, A. P., and Merrick, W. C. (1997) Biochim. Biophys. Acta 1350, 345 - 358）构建了真核延伸因子1A（以前称为eEF - 1α）的定点突变体。将核苷酸特异性序列NKMD（第153 - 156位氨基酸残基）作为诱变靶点，获得了以下突变体：N153D（DKMD）、N153T（TKMD）、D156N（NKMN）、D156W（NKMW）以及双突变体N153T,D156E（TKNE）。除了N153D突变体之外，所有以突变型eEF1A作为唯一eEF1A来源的酵母菌株都能存活。大多数纯化的突变型eEF1A在聚（U）指导的聚苯丙氨酸合成中的比活性与野生型相似，尽管其对GTP的Km值增加了1 - 2个数量级。这些突变体显示出GTP水解速率降低，并且大多数的错掺入率高于野生型。突变体NKMW eEF1A表现出不同寻常的特性。该酵母菌株对生长具有温度敏感性，尽管纯化后的蛋白质并非如此。其次，这种形式的eEF1A在蛋白质合成中的准确性比野生型高10倍，其GTP水解速率约为野生型的20%。总体而言，野生型蛋白质含有最优化的核苷酸特异性序列NKMD，即使该序列发生细微变化也会对eEF1A在体外的功能或酵母的生存能力产生重大影响。
