Mg2+ and a Key Lysine Modulate Exchange Activity of Eukaryotic Translation Elongation Factor 1Bα

• Published in: The Journal of biological chemistry 2006-07, Vol.281 (28), p.19457-19468
• Main Authors: Pittman, Yvette R., Valente, Louis, Jeppesen, Mads Gravers, Andersen, Gregers Rom, Patel, Smita, Kinzy, Terri Goss
• Format: Article
• Language: English
• Subjects: Catalysis; Eukaryotic Initiation Factor-1 - metabolism; Fungal Proteins - chemistry; Guanosine Diphosphate - chemistry; Kinetics; Lysine - chemistry; Magnesium - chemistry; Models, Molecular; Mutation; Nucleotides - chemistry; Peptide Elongation Factor 1 - chemistry; Peptide Elongation Factor 1 - genetics; Peptide Elongation Factors - chemistry; Protein Structure, Tertiary; Saccharomyces cerevisiae - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M601076200

To sustain efficient translation, eukaryotic elongation factor Bα (eEF1Bα) functions as the guanine nucleotide exchange factor for eEF1A. Stopped-flow kinetics using 2′-(or 3′)-O-N-methylanthraniloyl (mant)-GDP showed spontaneous release of nucleotide from eEF1A is extremely slow and accelerated 700-fold by eEF1Bα. The eEF1Bα-stimulated reaction was inhibited by Mg2+ with a K½ of 3.8 mm. Previous structural studies predicted the Lys-205 residue of eEF1Bα plays an important role in promoting nucleotide exchange by disrupting the Mg2+ binding site. Co-crystal structures of the lethal K205A mutant in the catalytic C terminus of eEF1Bα with eEF1A and eEF1A·GDP established that the lethality was not due to a structural defect. Instead, the K205A mutant drastically reduced the nucleotide exchange activity even at very low concentrations of Mg2+.A K205R eEF1Bα mutant on the other hand was functional in vivo and showed nearly wild-type nucleotide dissociation rates but almost no sensitivity to Mg2+. These results indicate the significant role of Mg2+ in the nucleotide exchange reaction by eEF1Bα and establish the catalytic function of Lys-205 in displacing Mg2+ from its binding site.