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 an d characterized. The amino acid replacement converted the guanine nucleotid e 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 pre sent in all the other GTP-binding proteins. The rate of poly(U)-directed po ly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacemen t partially affected the function of SsEF-2 during translocation. in contra st, the A26G substitution enhanced the catalytic efficiency of the intrinsi c SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scara no, G., & Bocchini, V. (1997) Biochimie 78, 832-837]. Surprisingly, A26GSsE F-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furth ermore, the alcohol increased the affinity for GTP without modifying the ca talytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [H-3]GDP was significantly reduced. These findings suggest t hat A26 is a regulator of the biochemical functions of SsEF-2. The involvem ent of this alanine residue in the guanine nucleotide-binding pocket of EF- 2 or EF-G is discussed.