Effect of propan-2-ol on enzymic and structural properties of elongation factor G

Abstract

ElongationfactorG (EF-G)can supporta GTPaseactivityinvitroeven intheabsenceofribosomeswhen propan-2-olispresent[GTPaseP;DeVendittis,Masullo& Bocchini(1986)J.Biol.Chem.261,4445-4450]. InthepresentworktheGTPasePactivityofEF-Gwas furtherstudiedbyinvestigating(i)theeffectofionic environmenton GTPasePand(i)theinfluenceofpropan-2-olon themolecularstructureofEF-Gas determinedbyfluorescenceandc.d.measurements. Inthepresence of1-300mm univalentcations(M+) alone,no detectableGTPasePactivitywas measured;however,inthepresence of1 mM-Mg2" a considerable stimulationwasobservedat40mM-Li'or75mM-NH4+.Amongbivalentcations(M2+),1 mM-Sr2+,2-5mM- Ca2'and1 mM-Ba2+were themosteffective,but,inthepresence of75mM-NH4+,Mg2+andMn2+became themosteficient,whereasthestimulationbyotherM2+specieswas considerablydecreased.C.d. measurementsshowedthatthealcoholincreasedthemean molarresidueelipticityofEF-Gat285nm, but notat220nm.Asestimatedfromfluorescencemeasurements,inthepresenceof20 (v/v)propan-2-olthe value of the dissociation constant of the complex formed between EF-G and 8-anilino-1 -naphthalene- sulphonatedecreasedfrom8to5/tM; similarly,thenumberofbindingsiteson EF-Gforthefluorescent 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 moderateconformationalchangesofEF-Gthatmakethecatalyticcentreaccessibletothesubstrateeven in the absence of ribosomes. Kinetics of GTPaseP studied at different temperatures did not reveal additional structuralchangesofEF-Goccurringwithtimeor temperature