GTP binding to elongation factor eEF-2 unmasks a tryptophan residue required for biological activity.
Elongation factor eEF-2 from rat liver, which contains 7 tryptophan residues, was treated with increasing concentrations of N-bromosuccinimide (NBS) under conditions in which these residues were oxidized specifically. The reagent produced a characteristic lowering in both the absorbance at 280 nm and the intrinsic fluorescence at 332 nm of the factor. Fluorometric titration of tryptophans and correlation to eEF-2 residual activity on GTP hydrolysis and polyphenylalanine synthesis showed that modification of the two most reactive tryptophans completely inactivated the factor. These residues were identified as Trp343 and Trp221 after cleavage of the protein with cyanogen bromide, separation of the fragments by reversed-phase high-pressure liquid chromatography, and N-terminal sequencing of the two fragments which exhibited a decreased absorbance in the NBS-treated protein. Oxidation of the most reactive residue, Trp343, did not induce significant decrease of activity of the factor or of its ability to interact with GTP or GDP. On the contrary, oxidation of Trp221 inactivated the factor, whose residual fluorescence was still partly quenched by GDP but no longer by GTP. Preincubation of eEF-2 with GDP protected Trp221 against NBS oxidation and prevented concomitant inactivation of the factor, whereas preincubation of eEF-2 with GTP increased the sensitivity of the same Trp221 residue to the reagent. Our results show for the first time that Trp221, which is conserved and belongs to a well preserved domain in eukaryotic cells and archaebacteria, plays an essential part in the catalytic activity of eEF-2. They strongly suggest that GTP induces a conformational change of the protein which unmasks this residue, whereas GDP stabilizes a conformation which makes this residue much less accessible.
Guillot
D
D
Laboratoires de Biochimie Médicale, Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique, Lyon, France.
Penin
F
F
Di Pietro
A
A
Sontag
B
B
Lavergne
J P
JP
Reboud
J P
JP
D000595:Amino Acid Sequence; D000818:Animals; D001974:Bromosuccinimide; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D001974:Bromosuccinimide