Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides. Export

@article{citeulike:5022123, abstract = {The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to 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 were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A F\"{o}rster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown.}, author = {Gonzalo, P. and Sontag, B. and Lavergne, J. P. and Jault, J. M. and Reboud, J. P.}, citeulike-article-id = {5022123}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/11063593}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=11063593}, issn = {0006-2960}, journal = {Biochemistry}, keywords = {atpase, ef-2}, month = {November}, number = {44}, pages = {13558--13564}, posted-at = {2009-06-30 13:35:46}, priority = {2}, title = {Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/11063593}, volume = {39}, year = {2000} }

TY - JOUR ID - citeulike:5022123 L3 - citeulike-article-id:5022123 N2 - The rat elongation factor eEF-2 catalyzes the translocation step of protein synthesis. Besides its well-characterized GTP/GDP binding properties, we have previously shown that ATP and ADP bind to 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 were different or not remained unclear. To further characterize these sites, eEF-2 was incubated in the presence of N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP. This led to an increase in the probe fluorescence and to a partial quenching of eEF-2 tryptophans in each case. The Mant-derivatives and the unmodified corresponding nucleotides were shown to bind to eEF-2 with a similar affinity. Competition experiments between Mant-labeled and unmodified nucleotides suggested the presence of two different sites binding either guanylic or adenylic nucleotides. A Förster's transfer between tryptophan residues and the Mant-probe is obtained with both the adenylic and the guanylic Mant-nucleotides, and comparison of the transfer efficiencies confirmed the presence of a second binding site specific for adenylic nucleotides. A sequence alignment of EF-Gs with eEF-2s from different species suggests the presence of potential Walker A and B motifs in an insert of the G-domain of eEF-2s from higher eukaryotes. Our results raise the possibility that a site specific for adenylic nucleotides and located in this insert has appeared in the course of evolution although its physiological function is still unknown. IS - 44 JF - Biochemistry SN - 0006-2960 EP - 13564 TI - Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides. VL - 39 SP - 13558 KW - atpase KW - ef-2 AU - Gonzalo, P AU - Sontag, B AU - Lavergne, JP AU - Jault, JM AU - Reboud, JP PY - 2000/11/07/ UR - http://view.ncbi.nlm.nih.gov/pubmed/11063593 ER -