Fasano O, Parmeggiani A
Biochemistry. 1981 Mar 3;20(5):1361-6. doi: 10.1021/bi00508a050.
In the preceding article a mutant elongation factor Tu (EF-TuD2216) resistant to the action of kirromycin was found to display a spontaneous guanosine 5'-triphosphatase (GTPase) activity, i.e., in the absence of aminoacyl transfer ribonucleic acid (tRNA) and ribosome-messenger RNA. This is the first example of an Ef-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. In this study we show that this activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation caused an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity an increased affinity for GTP are two properties also found with wild-type EF-Tu in the presence of kirromycin [Fasano, O., Burns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565; Sander, G., Okonek, M., Crechet, J.-B., Ivell, R., Bocchini, V., & Parmeggiani, A. (1979) FEBS Lett. 98, 111-114]. Therefore, both binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216.GDP but still does with EF-TuD2216.GTP in agreement with our previous finding that EF-Tu.GTP is the preferential target of the antibiotic in the wild type [Fasano, O., Bruns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565). The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome.mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(phenyl-alanine/leucine) synthesis at increasing Mg2+ concentrations, is identical for both wild-type and mutant EF-Tu.
在前一篇文章中，发现一种对奇霉素作用具有抗性的突变延伸因子Tu（EF-TuD2216）表现出一种自发的鸟苷5'-三磷酸酶（GTPase）活性，即在没有氨酰基转移核糖核酸（tRNA）和核糖体-信使核糖核酸（mRNA）的情况下。这是在没有大分子效应物和/或奇霉素的情况下，延伸因子Tu支持GTPase活性的第一个例子。在本研究中，我们表明这种活性是由增加铵离子浓度引发的。作为额外的效应，该突变导致EF-Tu对GTP的亲和力增加。GTPase活性对铵离子的依赖性以及对GTP亲和力的增加也是在存在奇霉素的情况下野生型EF-Tu所具有的两个特性[法萨诺，O.，伯恩斯，W.，克雷谢，J.-B.，桑德，G.，&帕尔梅贾尼，A.（1978年）《欧洲生物化学杂志》89卷，557 - 565页；桑德，G.，奥科内克，M.，克雷谢，J.-B.，伊韦尔，R.，博基尼，V.，&帕尔梅贾尼，A.（1979年）《欧洲生物化学学会联合会快报》98卷，111 - 114页]。因此，奇霉素与野生型EF-Tu的结合以及获得奇霉素抗性都引入了功能相关的修饰。高浓度（0.1 mM）的奇霉素不与突变体EF-TuD2216.GDP相互作用，但仍与EF-TuD2216.GTP相互作用，这与我们之前的发现一致，即EF-Tu.GTP是野生型中抗生素的优先作用靶点[法萨诺，O.，布伦斯，W.，克雷谢，J.-B.，桑德，G.，&帕尔梅贾尼，A.（1978年）《欧洲生物化学杂志》89卷，557 - 565页]。在存在氨酰基-tRNA和核糖体-mRNA的情况下，突变体EF-Tu的GTPase活性比野生型EF-Tu高得多，并且对mRNA存在的依赖性也小得多。在增加镁离子浓度时，以聚（U）指导的聚（苯丙氨酸/亮氨酸）合成来衡量的亮氨酸错义编码，野生型和突变体EF-Tu是相同的。
