Effect of propan-2-ol on enzymic and structural properties of elongation factor G.
Elongation factor G (EF-G) can support a GTPase activity in vitro even in the absence of ribosomes when propan-2-ol is present [GTPasep; De Vendittis, Masullo & Bocchini (1986) J. Biol. Chem. 261, 4445-4450]. In the present work the GTPasep activity of EF-G was further studied by investigating (i) the effect of ionic environment on GTPasep and (ii) the influence of propan-2-ol on the molecular structure of EF-G as determined by fluorescence and c.d. measurements. In the presence of 1-300 mM univalent cations (M+) alone, no detectable GTPasep activity was measured; however, in the presence of 1 mM-Mg2+ a considerable stimulation was observed at 40 mM-Li+ or 75 mM-NH4+. Among bivalent cations (M2+), 1 mM-Sr2+, 2-5 mM-Ca2+ and 1 mM-Ba2+ were the most effective, but, in the presence of 75 mM-NH4+, Mg2+ and Mn2+ became the most efficient, whereas the stimulation by other M2+ species was considerably decreased. C.d. measurements showed that the alcohol increased the mean molar residue ellipticity of EF-G at 285 nm, but not at 220 nm. As estimated from fluorescence measurements, in the presence of 20% (v/v) propan-2-ol the value of the dissociation constant of the complex formed between EF-G and 8-anilino-1-naphthalene-sulphonate decreased from 8 to 5 microM; similarly, the number of binding sites on EF-G for the fluorescent probe decreased from 13 to 6. Finally, the alcohol enhanced the quenching of the intrinsic fluorescence of EF-G caused by either acrylamide or KI. The data support the hypothesis that propan-2-ol induces moderate conformational changes of EF-G that make the catalytic centre accessible to the substrate even in the absence of ribosomes. Kinetics of GTPasep studied at different temperatures did not reveal additional structural changes of EF-G occurring with time or temperature.
Masullo
M
M
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli, Italy.
Parlato
G
G
De Vendittis
E
E
Bocchini
V
V
D000433:1-Propanol / Q000494:pharmacology*; D002942:Circular Dichroism; D005453:Fluorescence; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism*; D015394:Molecular Structure; D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D013696:Temperature
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D000433:1-Propanol; D010744:Phosphoric Monoester Hydrolases; D020963:GTP Phosphohydrolase-Linked Elongation Factors