G13A substitution affects the biochemical and physical properties of the elongation factor 1a. A reduced intrinsic GTPase is partially restored by kirromycin

The G13A substitution in the G13XXXXGK[T,S] consensus sequence of the elongation factor 1R from the archaeon Sulfolobus solfataricus (SsEF-1R) 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-1R wt), G13ASsEF-1R shows (i) a reduced rate of [3H]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 (GTPaseNa) [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376- 20379]. In addition, G13ASsEF-1R showed an increased affinity for GDP and GTP. Surprisingly, the decreased intrinsic GTPaseNa of G13ASsEF-1R can be partially restored by kirromycin, an effect not found for SsEF-1R wt. The temperature inducing a 50% denaturation of G13ASsEF-1R was somewhat lower (-5 °C) than that of SsEF-1R wt, and the decrease in its thermophilicity was slightly more accentuated (-10 °C). These results indicate that the nature of the residue in position 13 is important for the functional and physical properties of SsEF-1R.