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Evidence for a second nucleotide binding site in rat elongation factor eEF-2 specific for adenylic nucleotides
P Gonzalo1, B Sontag, J P Lavergne
1Laboratoire de Biochimie Médicale and Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, CNRS-UMR 5086, 7, passage du Vercors, 69367 Lyon Cedex 07, France. p.gonzalo@ibcp.fr
Biochemistry
|November 7, 2000
Summary
This study reveals that eukaryotic elongation factor 2 (eEF-2) has distinct binding sites for both guanine and adenine nucleotides, suggesting an evolutionary adaptation in protein synthesis.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Synthesis
Background:
- Eukaryotic elongation factor 2 (eEF-2) is crucial for protein synthesis translocation.
- Previous work indicated ATP/ADP binding to eEF-2, but distinct nucleotide-binding sites were unclear.
Purpose of the Study:
- To investigate whether eEF-2 possesses separate binding sites for guanine and adenine nucleotides.
- To characterize the nature and location of these nucleotide-binding sites.
Main Methods:
- Utilized N-methylanthraniloyl (Mant) fluorescent derivatives of GTP, GDP, ATP, and ADP.
- Performed fluorescence spectroscopy and Förster's resonance energy transfer (FRET) assays.
- Conducted competition experiments with unlabeled nucleotides.
Main Results:
- Mant-nucleotide derivatives bound eEF-2 with similar affinity to unlabeled nucleotides.
- Competition experiments indicated distinct binding sites for guanylic and adenylic nucleotides.
- FRET analysis confirmed a second, specific binding site for adenine nucleotides.
Conclusions:
- eEF-2 possesses separate binding sites for guanine and adenine nucleotides.
- A potential adenine nucleotide-binding site, possibly in a Walker motif-containing insert, may have evolved in higher eukaryotes.
- The physiological role of this specific adenine nucleotide-binding site remains to be elucidated.