Mg2+ Is Not Catalytically Required in the Intrinsic and Kirromycin-stimulated GTPase Action of Thermus thermophilus EF-Tu

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 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) ∼ no Me 2+ (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 (Kd ∼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 Me2+ < VO2+ < Mn 2+ ∼ Mg2+ whereas the binding affinity of GTP increased by one order of magnitude: no Me2+ < Mg2+ < VO2+