Valine114 Replacements In The Archaeal Elongation Factor 1a Enhanced Its Ability To Interact With Aminoacyl-Trna And Kirromycin(602 views) Masullo M, Cantiello P, Paola B, Fiengo A, Vitagliano L, Zagari A, Arcari P
Dipto. di Scienze Farmacobiologiche, Univ. di Catanzaro Magna Graecia, Roccelletta di Borgia, 1-88021 Catanzaro, Italy
Dipto. Biochimica e Biotecnol. Med., Universita di Napoli Federico II, CEINGE Biotecnologie Avanzate Scarl, via S. Pansini 5, 1-80131 Napoli, Italy
Ist. di Biostrutture e Bioimmagini, CNR, Departimento di Chimica Biologica, Via Mezzocannone 6, 1-80134 Napoli, Italy
References: Not available.
Valine114 Replacements In The Archaeal Elongation Factor 1a Enhanced Its Ability To Interact With Aminoacyl-Trna And Kirromycin
Valine 114 in the D109AAILVVA sequence of elongation factor 1α from the archaeon Sulfolobus solfataricus (SsEF-1α) was substituted with an acidic (V114E), basic (V114K), or cavity-forming (V114A) residue, and the effects on the biochemical properties of the factor were investigated. This sequence is well-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structure of SsEF-1α, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequence G13XXXXGK[T,S] [Vitagliano, L., Masullo, M., Sica, F., Zagari, A., and Bocchini, V. (2001) EMBO J. 20, 5305-5311]. These mutants displayed functions absent in the wild-type factor. In fact, although they exhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1α V114K, and V114A exhibited an affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than that elicited by SsEF-1α but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weaker binding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114E was drastically reduced compared to those of SsEF-1α, V114K, and V114A. Interestingly, the decreased intrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed for the G13A mutant of SsEF-1α [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., and Bocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginal effect on both the thermostability and thermophilicity of SsEF-1α, whereas V114K and V114E replacements strongly destabilized the molecule.
Valine114 Replacements In The Archaeal Elongation Factor 1a Enhanced Its Ability To Interact With Aminoacyl-Trna And Kirromycin