Sato K, Mori H, Yoshida M, Mizushima S
Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Nagatsuta 4259, Yokohama 226, Japan.
J Biol Chem. 1996 Jul 19;271(29):17439-44. doi: 10.1074/jbc.271.29.17439.
The high affinity ATP-binding site of SecA is located in its amino-terminal domain possessing amino acid sequences, the Walker A (GXXXXGKT) and B (ZZZZD) motifs, that are characteristic of a major class of nucleotide-binding sites (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951). Recently, we proposed that proteins possessing a typical set of Walker A and B motifs contain a conserved Glu or Asp between the two motifs. This Glu or Asp acts as a "catalytic residue" that activates a water molecule for an in-line attack on the gamma-phosphate of ATP (Amano, T., Yoshida, M., Matsuo, Y., and Nishikawa, K.(1995) FEBS Lett. 359, 1-5). In the present study, the aspartate residue at position 133 in Escherichia coli SecA, which could be the "catalytic residue," was mutated to an asparagine. The mutant SecA (SecA D133N) protein was expressed in E. coli CK4706, encoding a duplication of the secA gene, and purified to homogeneity. The in vitro protein translocation activity and membrane vesicle stimulated ATPase activity of SecA D133N were drastically reduced. Proteolytic studies indicated that the conformational changes of the mutant SecA occurring on interaction with ATP, presecretory proteins, phospholipids, and membrane vesicles, were similar to those of wild-type SecA. The mutant SecA allowed the signal peptide cleavage of proOmpA during translocation, indicating that the mutant retains the ability to bind ATP to perform the initial step of the translocation reaction. These data indicate that the carboxyl group of Asp-133 plays a role as a catalytic carboxylate, which activates a water molecule to attack gamma-phosphate of ATP, and the mutant lacking this residue cannot perform the total translocation but can still perform the initial step of the protein translocation.
SecA的高亲和力ATP结合位点位于其氨基末端结构域，该结构域拥有氨基酸序列，即沃克A基序（GXXXXGKT）和沃克B基序（ZZZZD），它们是一大类核苷酸结合位点的特征序列（沃克，J.E.，萨拉斯特，M.，伦斯威克，M.J.，和盖伊，N.J.（1982年）《欧洲分子生物学组织杂志》1，945 - 951）。最近，我们提出拥有一组典型沃克A和沃克B基序的蛋白质在这两个基序之间含有一个保守的谷氨酸或天冬氨酸。这个谷氨酸或天冬氨酸作为一个“催化残基”，激活一个水分子以对ATP的γ - 磷酸基团进行亲核攻击（天野，T.，吉田，M.，松尾，Y.，和西川，K.（1995年）《欧洲生物化学学会联合会快报》359，1 - 5）。在本研究中，大肠杆菌SecA中第133位的天冬氨酸残基，可能是“催化残基”，被突变为天冬酰胺。突变型SecA（SecA D133N）蛋白在编码secA基因重复序列的大肠杆菌CK4706中表达，并纯化至同质。SecA D133N的体外蛋白质转运活性和膜囊泡刺激的ATP酶活性大幅降低。蛋白水解研究表明，突变型SecA与ATP、分泌前蛋白、磷脂和膜囊泡相互作用时发生的构象变化与野生型SecA相似。突变型SecA在转运过程中允许前OmpA的信号肽切割，表明该突变体保留了结合ATP以进行转运反应初始步骤的能力。这些数据表明，Asp - 133的羧基作为催化羧酸盐发挥作用，激活一个水分子攻击ATP的γ - 磷酸基团，并且缺乏该残基的突变体不能完成整个转运过程，但仍能进行蛋白质转运的初始步骤。
