Valine 114 in the D109AAILVVA sequence of elongation factor 1R from the archaeon Sulfolobus solfataricus (SsEF-1R) 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-1R, 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-1R, 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-1R 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-1R, 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-1R [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-1R, whereas V114K and V114E replacements strongly destabilized the molecule.

Valine 114 Replacements in Archaeal Elongation Factor 1A Enhanced Its Ability To Interact with Aminoacyl-tRNA and Kirromycin

MASULLO, Mariorosario;
2002

Abstract

Valine 114 in the D109AAILVVA sequence of elongation factor 1R from the archaeon Sulfolobus solfataricus (SsEF-1R) 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-1R, 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-1R, 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-1R 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-1R, 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-1R [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-1R, whereas V114K and V114E replacements strongly destabilized the molecule.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11367/25740
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