Picha K M, Patel S S
Department of Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
J Biol Chem. 1998 Oct 16;273(42):27315-9. doi: 10.1074/jbc.273.42.27315.
The role of Mg2+ in dTTP hydrolysis, dTTP binding, hexamer formation, and DNA binding was studied in bacteriophage T7 DNA helicase (4A' protein). The steady state kcat for the dTTPase activity was 200-300-fold lower in the absence of MgCl2, but the Km was only slightly affected. Direct dTTP binding experiments showed that the Kd of dTTP was unaffected, but the stoichiometry of dTTP binding was different in the absence of Mg2+. Two dTTPs were found to bind tightly in the absence of Mg2+ in contrast to three to four in the presence of Mg2+. In the presence of DNA there was little difference in the stoichiometry of dTTP binding to 4A'. These results indicate that Mg2+ is not necessary for dTTP binding, but Mg2+ is required for optimal hydrolysis of dTTP. Gel filtration of 4A' in the presence of dTTP without Mg2+ showed that Mg2+ was not necessary, and dTTP was sufficient for hexamer formation. The hexamers formed in the presence of dTTP without Mg2+ were capable of binding single-stranded DNA. However, the 4A' hexamers formed in the presence of dTDP with or without Mg2+ did not bind DNA, indicating that hexamer formation itself is not sufficient for DNA binding. The hexamers need to be in the correct conformation, in this case in the dTTP-bound state, to interact with the DNA. Thus, the gamma-phosphate of dTTP plays an important role in causing a conformational change in the protein that leads to stable interactions of 4A' with the DNA.
研究了镁离子（Mg2+）在噬菌体T7 DNA解旋酶（4A'蛋白）的dTTP水解、dTTP结合、六聚体形成及DNA结合过程中的作用。在没有MgCl2的情况下，dTTPase活性的稳态催化常数（kcat）降低了200 - 300倍，但米氏常数（Km）仅受到轻微影响。直接的dTTP结合实验表明，dTTP的解离常数（Kd）不受影响，但在没有Mg2+时，dTTP结合的化学计量不同。结果发现，在没有Mg2+时两个dTTP紧密结合，而在有Mg2+时则为三到四个。在有DNA存在的情况下，dTTP与4A'结合的化学计量几乎没有差异。这些结果表明，Mg2+对于dTTP结合并非必需，但对于dTTP的最佳水解是必需的。在没有Mg2+的情况下对存在dTTP的4A'进行凝胶过滤显示，Mg2+并非必需，dTTP足以形成六聚体。在没有Mg2+的情况下由dTTP存在时形成的六聚体能够结合单链DNA。然而，在有或没有Mg2+的情况下由dTDP存在时形成的4A'六聚体不结合DNA，这表明六聚体的形成本身不足以结合DNA。六聚体需要处于正确的构象，在这种情况下是处于dTTP结合状态，才能与DNA相互作用。因此，dTTP的γ-磷酸在引起蛋白质构象变化中起重要作用，这种构象变化导致4A'与DNA的稳定相互作用。
