Evidence for a Second Nucleotide Binding Site in Rat Elongation Factor eEF-2 Specific for Adenylic Nucleotides

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• 作者：Philippe Gonzalo ; Bruno Sontag ; Jean-Pierre Lavergne ; Jean-Michel Jault ; and Jean-Paul Reboud
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：November 7, 2000
• 年：2000
• 卷：39
• 期：44
• 页码：13558 - 13564
• 全文大小：164K
• 年卷期：v.39,no.44(November 7, 2000)
• ISSN：1520-4995

文摘

The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besidesits well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bindto eEF-2 [Sontag, B., Reboud, A. M., Divita, G., Di Pietro, A., Guillot, D., and Reboud, J. P. (1993)Biochemistry 32, 1976-1980]. However, whether the adenylic and guanylic nucleotide binding sites weredifferent or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presenceof N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to anincrease in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. TheMant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with asimilar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggestedthe presence of two different sites binding either guanylic or adenylic nucleotides. A Förster's transferbetween tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylicMant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second bindingsite specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different speciessuggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s fromhigher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and locatedin this insert has appeared in the course of evolution although its physiological function is still unknown.