G13A substitution affects the biochemical and physical properties of the elongation factor 1 alpha. A reduced intrinsic GTPase activity is partially restored by kirromycin.
The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1 alpha from the archaeon Sulfolobus solfataricus (SsEF-1 alpha) was introduced in order to study the reasons for selective differences found in the homologous consensus element AXXXXGK[T,S] of the other elongation factor EF-2 or EF-G. In a previous work, it was shown that the main effect of the A26G mutation was the activation of the intrinsic GTPase of SsEF-2 [De Vendittis, E., Adinolfi, B. S., Amatruda, M. R., Raimo, G., Masullo, M., and Bocchini, V. (1994) Eur. J. Biochem. 262, 600-605]. In this work, we found that, compared to the wild-type factor (SsEF-1 alpha wt), G13ASsEF-1 alpha shows (i) a reduced rate of [(3)H]Phe polymerization that was probably due to its reduced ability to form a ternary complex with heterologous aa-tRNA and (ii) a reduced intrinsic GTPase activity that was stimulated by high concentrations of NaCl (GTPase(Na)) [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. In addition, G13ASsEF-1 alpha showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPase(Na) of G13ASsEF-1 alpha can be partially restored by kirromycin, an effect not found for SsEF-1 alpha wt. The temperature inducing a 50% denaturation of G13ASsEF-1 alpha was somewhat lower (-5 degrees C) than that of SsEF-1 alpha wt, and the decrease in its thermophilicity was slightly more accentuated (-10 degrees C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1 alpha.
Masullo
Mariorosario
M
Dipartimento di Scienze Farmacobiologiche, Università degli Studi di Catanzaro Magna Graecia, Complesso Ninì Barbieri, Roccelletta di Borgia, I-88021 Catanzaro, Italy.
Cantiello
Piergiuseppe
P
de Paola
Barbara
B
Catanzano
Francesca
F
Arcari
Paolo
P
Bocchini
Vincenzo
V
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000378:metabolism*; D006147:Guanine / Q000737:chemistry; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006358:Hot Temperature; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020648:Peptide Elongation Factor 1 / Q000737:chemistry* / Q000502:physiology; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011728:Pyridones / Q000494:pharmacology*; D016944:Sulfolobus / Q000201:enzymology; D013696:Temperature; D013997:Time Factors
D000900:Anti-Bacterial Agents; D020648:Peptide Elongation Factor 1; D011728:Pyridones; D006153:Guanosine Diphosphate; D006147:Guanine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; C008536:mocimycin