Open AccessThe A26G replacement in the consensus sequence A‐X‐X‐X‐X‐G‐K‐[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon Sulfolobus solfataricus
Author(s) -
De Vendittis Emmanuele,
Adinolfi Bianca Stella,
Amatruda Maria Rosa,
Raimo Gennaro,
Masullo Mariorosario,
Bocchini Vincenzo
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00428.x
Subject(s) - gtpase , gtp' , nucleotide , ef tu , guanine , sulfolobus solfataricus , stereochemistry , chemistry , elongation factor , g protein , biochemistry , biology , enzyme , transfer rna , ribosome , receptor , rna , archaea , gene
A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus ( Ss EF‐2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A‐X‐X‐X‐X‐G‐K‐[T,S] of the elongation factors EF‐G or EF‐2 into the corresponding G‐X‐X‐X‐X‐G‐K‐[T,S] motif which is present in all the other GTP‐binding proteins. The rate of poly(U)‐directed poly(Phe) synthesis and the ribosome‐dependent GTPase activity of A26G Ss EF‐2 were decreased compared to Ss EF‐2, thus indicating that the A26G replacement partially affected the function of Ss EF‐2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic Ss EF‐2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78 , 832–837]. Surprisingly, A26G Ss EF‐2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26G Ss EF‐2 GTPase. Compared to Ss EF‐2, the affinity of A26G Ss EF‐2 for [ 3 H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of Ss EF‐2. The involvement of this alanine residue in the guanine nucleotide‐binding pocket of EF‐2 or EF‐G is discussed.