The 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.
A recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-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 A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsEF-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 A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide-binding pocket of EF-2 or EF-G is discussed.
De Vendittis
E
E
Dipartimento dei Biochimica e Biotecnologie Mediche, Università di Napoli Frederico, II, Italy.
Adinolfi
B S
BS
Amatruda
M R
MR
Raimo
G
G
Masullo
M
M
Bocchini
V
V
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D016944:Sulfolobus / Q000201:enzymology / Q000378:metabolism*
D017931:DNA Primers; D006150:Guanine Nucleotides; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D020963:GTP Phosphohydrolase-Linked Elongation Factors