1,721,115 research outputs found
G13A substitution affects the biochemical and physical properties of the elongation factor 1a. A reduced intrinsic GTPase is partially restored by kirromycin
No full textThe 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
The A26G replacement in the consensus sequence A-X-X-X-X-G-K-[T,S] of the guanine nucleotide binding site activates the intrinsic GTPase of the elongation factor 2 from the archaeon sulfolobus solfataricus
No full textA recombinant form of the elongation factor 2 from the archaeon Sulfolobus solfataricus (SsEF-2), carrying the A26G substitution, has been produced and characterized. The amino acid replacement converted the guanine nucleotide binding consensus sequences A-X-X-X-X-G-K-[T,S] of the elongation factors EF-G or EF-2 into the corresponding G-X-X-X-X-G-K-[T,S] motif which is present in all the other GTP-binding proteins. The rate of poly(U)-directed poly(Phe) synthesis and the ribosome-dependent GTPase activity of A26GSsEF-2 were decreased compared to SsEF-2, thus indicating that the A26G replacement partially affected the function of SsEF-2 during translocation. In contrast, the A26G substitution enhanced the catalytic efficiency of the intrinsic SsEF-2 GTPase triggered by ethylene glycol [Raimo, G., Masullo, M., Scarano, G., & Bocchini, V. (1997) Biochimie 78, 832±837]. Surprisingly, A26GSsEF-2 was able to hydrolyse GTP even in the absence of ethylene glycol; furthermore, the alcohol increased the affinity for GTP without modifying the catalytic constant of A26GSsEF-2 GTPase. Compared to SsEF-2, the affinity of A26GSsEF-2 for [3H]GDP was significantly reduced. These findings suggest that A26 is a regulator of the biochemical functions of SsEF-2. The involvement of this alanine residue in the guanine nucleotide- binding pocket of EF-2 or EF-G is discussed
Salts induce structural changes in elongation factor 1a from the hyperthermophilic archaeon Sulfolobus solfataricus: a Fourier transform infrared spectroscopy study
No full textElongation factor 1R from the hyperthermophilic archaeon Sulfolobus solfataricus (SsEF-1R) carries the aminoacyl tRNA to the ribosome; it binds GDP or GTP, and it is also endowed with an intrinsic GTPase activity that is triggered in vitro by NaCl at molar concentrations [Masullo, M., De Vendittis, E., and Bocchini, V. (1994) J. Biol. Chem. 269, 20376-20379]. The structural properties of SsEF-1R were investigated by Fourier transform infrared spectroscopy. The estimation of the secondary structure of the SsEF-1R‚GDP complex, made by curve fitting of the amide I′ band or by factor analysis of the amide I band, indicated a content of 34-36% R-helix, 35-40% -sheet, 14-19% turn, and 7% unordered structure. The substitution of the GDP bound with the slowly hydrolyzable GTP analogue Gpp(NH)p induced a slight increase in the R-helix and -sheet content. On the other hand, the R-helix content of the SsEF- 1R‚GDP complex increased upon addition of salts, and the highest effect was produced by 5 M NaCl. The thermal stability of the SsEF-1R‚GDP complex was significantly reduced when the GDP was replaced with Gpp(NH)p or in the presence of NaBr or NH4Cl, whereas a lower destabilizing effect was provoked by NaCl and KCl. Therefore, the extent of the destabilizing effect of salts depended on the nature of both the cation and the anion. The data suggested that the sodium ion was responsible for the induction of the GTPase activity, whereas the anion modulated the enzymatic activity through destabilization of particular regions of SsEF-1R. Finally, the infrared data suggested that, in particular region(s) of the polypeptide chain, the SsEF-1R‚Gpp(NH)p complex possesses structural conformations which are different from those present in the SsEF-1R‚GDP complex
Codon usage and amino acid composition in sulfothermophiles. An approach for the understanding of protein adaptation to unusual growth environments
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Codon usage and amino acid composition in sulfothermophiles. An approach for the understanding of protein adaptation to unusual growth environments
No full textThe elongation factor G carries a catalytic site for GTP hydrolysis, which is revealed by using 2-propanol in the absence of ribosomes
No full textIn the absence of ribosomal particles, elongation factor G (EF-G) promotes very little GTP hydrolysis. After the addition of some aliphatic alcohols to EF-G, the rate of nucleotide cleavage was significantly increased and GTPase activity was easily detectable. The highest stimulation, nearly 16-fold, occurred with 2-propanol at a 20% (v/v) concentration. The reaction showed the characteristics of an enzymatic catalysis, but the rate was three orders of magnitude lower than that of the ribosome-dependent EF-G GTPase activity. Striking similarities between the two activities indicated that the catalysis stimulated by the alcohol was due to EF-G itself. We found that EF-G GTPase activity in the presence of 2-propanol displayed an absolute specificity for GTP as in the presence of ribosomes; the two activities copurified to a constant ratio and exhibited coincident chromatographic and electrophoretic patterns; the temperature for the half-inactivation of EF-G was 59.3 degrees C for both GTPase systems, as well as the kinetic constant for the thermal inactivation process which was found to be 0.05 min-1; and the Km for the GTP in the presence of 2-propanol (59 microM) was similar to that found in the presence of ribosomes. These results indicate that the EF-G molecule carries a catalytic site for GTP hydrolysis, which in the absence of ribosomal particles is activated by an appropriate alcohol/water surrounding medium
Molecular and functional properties of an archaeal phenylalanyl synthetase from the hyperthermophile Sulfolobus solfataricus
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