Biochemical characterisation of the D60A mutant of the elongation factor 1alpha from the archaeon Sulfolobus solfataricus.
The D60A mutant of the elongation factor (EF) 1alpha from Sulfolobus solfataricus (Ss), was obtained as heterologous expressed protein and characterised. This substitution was carried out in order to analyse the involvement of this evolutionally conserved amino acid position in the interaction between the elongation factor and guanosine nucleotides and in the coordination of magnesium ions. The expression system used produced a folded protein able to catalyse, although to a slightly lower extent with respect to the wild-type enzyme, protein synthesis in vitro and NaCl-dependent intrinsic GTPase activity. The affinity for guanosine nucleotides was almost identical to that exhibited by wild-type SsEF-1alpha; vice versa, the GDP exchange rate was one order of magnitude faster on the mutated elongation factor, a property partially restored when the exchange reaction was analysed in the presence of the magnesium ions chelating agent EDTA. Finally, the D60A substitution only a little affected the high thermal stability of the elongation factor. From a structural point of view, the analysis of the data reported confirmed that this conserved carboxyl group belongs to a protein region differentiating the GDP binding mode among elongation factors from different organisms.
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Via S. Pansini 5, I-80131 Napoli, Italy.
Immacolata
I
Ruggiero
Piergiuseppe
P
Cantiello
Anna
A
Lamberti
Angela
A
Sorrentino
Nicola M
NM
Martucci
Alessia
A
Ruggiero
Rosaria
R
Arcone
Luigi
L
Vitagliano
Paolo
P
Arcari
Mariorosario
M
Masullo
D005822:Genetic Vectors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D048229:Sulfolobus solfataricus / Q000378:metabolism*
D020648:Peptide Elongation Factor 1; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate