Pittman Yvette R, Valente Louis, Jeppesen Mads Gravers, Andersen Gregers Rom, Patel Smita, Kinzy Terri Goss
Department of Molecular Genetics, Microbiology & Immunology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854, USA.
J Biol Chem. 2006 Jul 14;281(28):19457-68. doi: 10.1074/jbc.M601076200. Epub 2006 May 4.
To sustain efficient translation, eukaryotic elongation factor B alpha (eEF1B alpha) functions as the guanine nucleotide exchange factor for eEF1A. Stopped-flow kinetics using 2'-(or 3')-O-N-methylanthraniloyl (mant)-GDP showed spontaneous release of nucleotide from eEF1A is extremely slow and accelerated 700-fold by eEF1B alpha. The eEF1B alpha-stimulated reaction was inhibited by Mg2+ with a K(1/2) of 3.8 mM. Previous structural studies predicted the Lys-205 residue of eEF1B alpha plays an important role in promoting nucleotide exchange by disrupting the Mg2+ binding site. Co-crystal structures of the lethal K205A mutant in the catalytic C terminus of eEF1B alpha with eEF1A and eEF1A.GDP established that the lethality was not due to a structural defect. Instead, the K205A mutant drastically reduced the nucleotide exchange activity even at very low concentrations of Mg2+. A K205R eEF1B alpha mutant on the other hand was functional in vivo and showed nearly wild-type nucleotide dissociation rates but almost no sensitivity to Mg2+. These results indicate the significant role of Mg2+ in the nucleotide exchange reaction by eEF1B alpha and establish the catalytic function of Lys-205 in displacing Mg2+ from its binding site.
为维持高效翻译，真核生物延伸因子Bα（eEF1Bα）作为eEF1A的鸟嘌呤核苷酸交换因子发挥作用。使用2'-（或3'）-O-N-甲基邻氨基苯甲酰（mant）-GDP的停流动力学表明，eEF1A中核苷酸的自发释放极其缓慢，而eEF1Bα可使其加速700倍。eEF1Bα刺激的反应受到Mg2+的抑制，其K(1/2)为3.8 mM。先前的结构研究预测，eEF1Bα的Lys-205残基通过破坏Mg +结合位点在促进核苷酸交换中起重要作用。eEF1Bα催化C末端的致死性K205A突变体与eEF1A和eEF1A.GDP的共晶体结构表明，致死性并非由于结构缺陷。相反，K205A突变体即使在非常低的Mg2+浓度下也会大幅降低核苷酸交换活性。另一方面，K205R eEF1Bα突变体在体内具有功能，显示出几乎野生型的核苷酸解离速率，但对Mg2+几乎没有敏感性。这些结果表明Mg2+在eEF1Bα介导的核苷酸交换反应中的重要作用，并确立了Lys-205在将Mg2+从其结合位点置换中的催化功能。
