Mg2+ is not catalytically required in the intrinsic and kirromycin-stimulated GTPase action of Thermus thermophilus EF-Tu.
The influence of divalent metal ions on the intrinsic and kirromycin-stimulated GTPase activity in the absence of programmed ribosomes and on nucleotide binding affinity of elongation factor Tu (EF-Tu) from Thermus thermophilus prepared as the nucleotide- and Mg(2+)-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn(2+) (0.069 min(-1)) > Mg(2+) (0.037 min(-1)) approximately no Me(2+) (0.034 min(-1)) > VO(2+) (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg(2+) retarded the rate of hydrolysis in comparison to that in the absence of divalent metal ions, an effect ascribed to kinetics of nucleotide exchange. In the absence of added divalent metal ions, GDP and GTP were bound with equal affinity (K(d) approximately 10(-7) m). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me(2+) < VO(2+) < Mn(2+) approximately Mg(2+) whereas the binding affinity of GTP increased by one order of magnitude: no Me(2+) < Mg(2+) < VO(2+) < Mn(2+). Estimates of equilibrium (dissociation) binding constants for GDP and GTP by EF-Tu on the basis of Scatchard plot analysis, together with thermodynamic data for hydrolysis of triphosphate nucleotides (Phillips, R. C., George, P., and Rutman, R. J. (1969) J. Biol. Chem. 244, 3330-3342), showed that divalent metal ions stabilize the EF-Tu.Me(2+).GDP complex over the protein-free Me(2+).GDP complex in solution, with the effect greatest in the presence of Mg(2+) by approximately 10 kJ/mol. These combined results show that Mg(2+) is not a catalytically obligatory cofactor in intrinsic and kirromycin-stimulated GTPase action of EF-Tu in the absence of programmed ribosomes, which highlights the differential role of Mg(2+) in EF-Tu function.
Laboratorium für Biochemie, Universität Bayreuth, 95440 Bayreuth, Germany.
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Rutthard
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Banerjee
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Makinen
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism*; D008956:Models, Chemical; D009711:Nucleotides / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D011485:Protein Binding; D011728:Pyridones / Q000494:pharmacology*; D013057:Spectrum Analysis; D013816:Thermodynamics; D016963:Thermus thermophilus / Q000378:metabolism*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D007477:Ions; D009711:Nucleotides; D011728:Pyridones; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D010444:Peptide Elongation Factor Tu; D008274:Magnesium; C008536:mocimycin