Abstract
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.
Highlights
The GTPase superfamily of proteins, known more commonly as G-proteins, are ubiquitous in cellular systems and serve as key regulatory molecules catalyzing the hydrolysis of the ,␥phosphate bond in GTP [1,2,3,4]
Removal of Mg2ϩ—In a variety of studies, the influence of divalent metal ions on the intrinsic, kirromycin-stimulated, and physiological GTPase activity of elongation factor Tu (EF-Tu) has been examined for EF-Tu of both E. coli [37, 38] and thermophilic [19, 20, 24] organisms
The influence of monovalent and divalent metal ions on the intrinsic GTPase activity of p21ras has been compared with that of EF-Tu isolated from E. coli [39]
Summary
The GTPase superfamily of proteins, known more commonly as G-proteins, are ubiquitous in cellular systems and serve as key regulatory molecules catalyzing the hydrolysis of the ,␥phosphate bond in GTP [1,2,3,4]. In contrast to the structural instability of elongation factor Tu of mesophiles such as E. coli, the homologous thermostable protein in its nucleotide-free form is less prone to inactivation, and comparison of the kinetics of nucleotide binding shows it to be identical to EF-Tu isolated as the GDP-bound complex from the cytosol [11, 16] This characteristic has allowed application of efficient biochemical purification methods for preparation of the nucleotide-free elongation factor from Thermus thermophilus [16, 17] and Bacillus stearothermophilus [18, 19] for x-ray structure analysis of the complex formed with an inhibitor analog of GTP in the active site [5, 6]. The differential role of Mg2ϩ in EF-Tu function is discussed further through analysis of thermodynamic relationships governing the hydrolysis of nucleoside 5Ј-triphosphates [27, 28] and the kinetic requirement for a nucleotide-exchange factor in the cell
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