Valine 114 Replacements in Archaeal Elongation Factor 1 Enhanced Its Ability To Interact with Aminoacyl-tRNA and Kirromycin
详细信息 查看全文
- 作者：Mariorosario Masullo ; Piergiuseppe Cantiello ; Barbara de Paola ; Antonio Fiengo ; Luigi Vitagliano ; Adriana Zagari ; and Paolo Arcari
- 刊名：Biochemistry
- 出版年：2002
- 出版时间：December 10, 2002
- 年：2002
- 卷：41
- 期：49
- 页码：14482 - 14488
- 全文大小：191K
- 年卷期：v.41,no.49(December 10, 2002)
- ISSN：1520-4995
文摘
Valine 114 in the D109AAILVVA sequence of elongation factor 1
from the archaeon Sulfolobussolfataricus (SsEF-1) 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 iswell-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structureof SsEF-1, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequenceG13XXXXGK[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 theyexhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1, V114K and V114A exhibitedan affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than thatelicited by SsEF-1 but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weakerbinding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114Ewas drastically reduced compared to those of SsEF-1, V114K, and V114A. Interestingly, the decreasedintrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed forthe G13A mutant of SsEF-1 [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., andBocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginaleffect on both the thermostability and thermophilicity of SsEF-1, whereas V114K and V114E replacementsstrongly destabilized the molecule.
from the archaeon Sulfolobussolfataricus (SsEF-1) 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 iswell-conserved among most of eukaryal and eubacterial counterparts, and in the three-dimensional structureof SsEF-1, V114 is located in a hydrophobic pocket near the first GDP-binding consensus sequenceG13XXXXGK[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 theyexhibited a rate in poly(Phe) incorporation almost identical to that of SsEF-1, V114K and V114A exhibitedan affinity for GDP and GTP higher and a capability to bind heterologous aa-tRNA stronger than thatelicited by SsEF-1 but similar to that of eubacterial EF-Tu. V114E instead displayed not only a weakerbinding capability for aa-tRNA but also a lower affinity for GDP. The intrinsic GTPase activity of V114Ewas drastically reduced compared to those of SsEF-1, V114K, and V114A. Interestingly, the decreasedintrinsic GTPase activity of V114E was partially restored by kirromycin, an effect already observed forthe G13A mutant of SsEF-1 [Masullo, M., Cantiello, P., de Paola, B., Catanzano, F., Arcari, P., andBocchini, V. (2002) Biochemistry 41, 628-633]. Finally, the V114A substitution showed only a marginaleffect on both the thermostability and thermophilicity of SsEF-1, whereas V114K and V114E replacementsstrongly destabilized the molecule.