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 Mg2+-free protein has been investigated. The intrinsic GTPase activity under single turnover conditions varied according to the series: Mn2+ (0.069 min(-1)) > Mg2+ (0.037 min(-1)) similar to no Me2+ (0.034 min(-1)) > VO2+ (0.014 min(-1)). The kirromycin-stimulated activity showed a parallel variation. Under multiple turnover conditions (GTP/EF-Tu ratio of 10:1), Mg2+ 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 (R, similar to 10(-7) hr). In the presence of added divalent metal ions, GDP affinity increased by up to two orders of magnitude according to the series: no Me2+ < VO2+ < Mn2+ similar to Mg2+ whereas the binding affinity of G;TP increased by one order of magnitude: no Me2+ < Mg2+ < VO2+ < Mn2+ 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 . Me2+ GDP complex over the protein-free Me2+ GDP complex in solution, with the effect greatest in the presence of Mg2+ by similar to 10 kJ/mol. These combined results show that Mg2+ 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 Mg2+ in EF-Tu function.