Anborgh P H, Parmeggiani A
S.D.I. no. 61840 du Centre National de la Recherche Scientifique, Laboratoire de Biochimie, Ecole Polytechnique, Palaiseau, France.
J Biol Chem. 1993 Nov 25;268(33):24622-8.
The activity of Escherichia coli elongation factor Tu (EF-Tu) depends on its GTP- and GDP-bound conformations. In this work we have studied the influence of the antibiotic GE2270 A, a new EF-Tu-specific inhibitor of protein biosynthesis, on these two conformations with respect to the interaction with the various ligands and stability. One molecule of GE2270 A bound per EF-Tu is sufficient to block poly(U)-directed poly(Phe) incorporation. This drug binds stably to both EF-Tu.GTP and EF-Tu.GDP but only affects the reactivity of the GTP-bound conformation that is no longer able to interact efficiently with aminoacyl-tRNA (aa-tRNA) and ribosomes. Consequently, the protection by EF-Tu.GTP of the aminoacyl-ester bond against nonenzymatic hydrolysis is strongly weakened. The intrinsic EF-Tu GTPase is little affected, but its response to aa-tRNA and ribosomes is impaired. The specificity of GE2270 A to the GTP-bound form of EF-Tu is supported by the course of temperature- and urea-dependent inactivation or tryptic digestion. Furthermore, in its presence elongation factor Ts (EF-Ts) can enhance the dissociation of EF-Tu.GDP but not that of EF-Tu.GTP. Unlike kirromycin, this antibiotic can bind to EF-Tu.EF-Ts without causing dissociation of this complex. Evidence was obtained that the EF-Tu binding site for GE2270 A is also distinct from those for kirromycin, pulvomycin, and aa-tRNA, even though this antibiotic functionally interferes with all three of these ligands. GE2270 A appears to interact with a crucial regulatory region of the EF-Tu molecule that is activated in the GTP-bound state.
大肠杆菌延伸因子Tu（EF-Tu）的活性取决于其结合GTP和GDP的构象。在本研究中，我们研究了抗生素GE2270 A（一种新型的蛋白质生物合成中特异性抑制EF-Tu的抑制剂）对这两种构象在与各种配体相互作用及稳定性方面的影响。每个EF-Tu结合一个GE2270 A分子就足以阻断聚（U）指导的聚（苯丙氨酸）掺入。这种药物能稳定地结合到EF-Tu·GTP和EF-Tu·GDP上，但仅影响结合GTP构象的反应性，该构象不再能够与氨酰-tRNA（aa-tRNA）和核糖体有效相互作用。因此，EF-Tu·GTP对氨酰酯键的非酶促水解的保护作用被大大削弱。内在的EF-Tu GTP酶受影响较小，但其对aa-tRNA和核糖体的反应受损。温度和尿素依赖性失活或胰蛋白酶消化过程支持了GE2270 A对EF-Tu的GTP结合形式的特异性。此外，在其存在下，延伸因子Ts（EF-Ts）可以增强EF-Tu·GDP的解离，但不能增强EF-Tu·GTP的解离。与奇霉素不同，这种抗生素可以结合到EF-Tu·EF-Ts上而不导致该复合物解离。有证据表明，GE2270 A的EF-Tu结合位点也与奇霉素、普尔霉素和aa-tRNA的结合位点不同，尽管这种抗生素在功能上干扰了所有这三种配体。GE2270 A似乎与EF-Tu分子中在GTP结合状态下被激活的关键调节区域相互作用。
