Wieden Hans-Joachim, Gromadski Kirill, Rodnin Dmytro, Rodnina Marina V
Institute of Physical Biochemistry, University of Witten/Herdecke, 58448 Witten, Germany.
J Biol Chem. 2002 Feb 22;277(8):6032-6. doi: 10.1074/jbc.M110888200. Epub 2001 Dec 13.
Nucleotide exchange in elongation factor Tu (EF-Tu) is catalyzed by elongation factor Ts (EF-Ts). Similarly to other GTP-binding proteins, the structural changes in the P loop and the Mg(2+) binding site are known to be important for nucleotide release from EF-Tu. In the present paper, we determine the contribution of the contacts between helix D of EF-Tu at the base side of the nucleotide and the N-terminal domain of EF-Ts to the catalysis. The rate constants of the multistep reaction between Escherichia coli EF-Tu, EF-Ts, and GDP were determined by stopped-flow kinetic analysis monitoring the fluorescence of either Trp-184 in EF-Tu or mant-GDP. Mutational analysis shows that contacts between helix D of EF-Tu and the N-terminal domain of EF-Ts are important for both complex formation and the acceleration of GDP dissociation. The kinetic results suggest that the initial contact of EF-Ts with helix D of EF-Tu weakens binding interactions around the guanine base, whereas contacts of EF-Ts with the phosphate binding side that promotes the release of the phosphate moiety of GDP appear to take place later. This "base-side-first" mechanism of guanine nucleotide release resembles that found for Ran x RCC1 and differs from mechanisms described for other GTPase x GEF complexes where interactions at the phosphate side of the nucleotide are released first.
延伸因子Ts（EF-Ts）催化延伸因子Tu（EF-Tu）中的核苷酸交换。与其他GTP结合蛋白类似，已知P环和Mg(2+)结合位点的结构变化对于核苷酸从EF-Tu释放很重要。在本文中，我们确定了EF-Tu核苷酸碱基侧的螺旋D与EF-Ts的N端结构域之间的接触对催化作用的贡献。通过监测EF-Tu中Trp-184或mant-GDP的荧光的停流动力学分析，测定了大肠杆菌EF-Tu、EF-Ts和GDP之间多步反应的速率常数。突变分析表明，EF-Tu的螺旋D与EF-Ts的N端结构域之间的接触对于复合物形成和GDP解离的加速都很重要。动力学结果表明，EF-Ts与EF-Tu的螺旋D的初始接触会削弱鸟嘌呤碱基周围的结合相互作用，而EF-Ts与促进GDP磷酸基团释放的磷酸结合侧的接触似乎稍后发生。这种鸟嘌呤核苷酸释放的“碱基侧优先”机制类似于在Ran x RCC1中发现的机制，与其他GTPase x GEF复合物中首先释放核苷酸磷酸侧相互作用的机制不同。
