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 Mg 2؉ -free protein has been investigated. 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 (1-4). For most G-proteins this process yields a tightly bound enzyme-product complex requiring the action of a nucleotide exchange factor to kinetically facilitate exchange of the GTP for GDP in the active site. Nucleotide binding and hydrolysis are key to regulating the activity of these enzymes, and both of these steps occur in the presence of the divalent metal ion Mg 2ϩ as the naturally occurring cofactor in the cell. However, in EF-Tu 1 function it has not been possible hitherto to establish whether there is a differential role of the divalent metal ion in nucleotide hydrolysis as opposed to nucleotide binding.In all G-proteins defined hitherto by x-ray crystallographic studies, the substrate analog guanosine 5Ј-(␤,␥-imido)-triphosphate is bound as a complex with Mg 2ϩ in which the divalent metal ion is coordinated to oxygen atoms from the ␤-phosphate and ␥-phosphate groups, and the guanine and divalent metal ion binding sites appear to be tightly coupled (5, 6). For ras (7), for elongation factor Tu from both thermophilic bacteria (8 -11) and Escherichia coli (12, 13), and for G␣-subunits of heterotrimeric G-proteins (14, 15), the dissociation constants for release of protein-bound nucleotide measured in the presence of Mg 2ϩ indicate differential binding affinities for GDP and GTP by at least an order of magnitude. Binding affinities for both nucleotides are often measured kinetically by ligand displacement because G-proteins are less stable when prepared in their nucleotide-free form. Because nucleotide separation is necessary for complete removal of Mg 2ϩ from the protein, the less stable, nucleotide-free form of G-proteins complicates efforts to determine whether the divalent metal ion has differential roles in nucleotide hydrolysis and nucleotide binding.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). Isolation of nucleotidefree elongation factor Tu (17-20) and p21 ras (7) has also allowed comp...

Riboneogenesis in Yeast

“…In addition, the Shb17-SBP structure revealed the presence of an additional density close to the side chains of the conserved T16 and T25 (2.2 Å and 3.2 Å) and to the P1 oxygen (2.9 Å), which was interpreted as a Mg 2+ cation (Figure 3B). This type of Mg 2+ coordination is very similar to that found in the regulatory and dynamin-like GTPases/ATPases, where Mg 2+ is not required for substrate hydrolysis, but contributes to the phosphate coordination (Daumke et al, 2007; Rutthard et al, 2001; Zhang et al, 2000). There is a similar density at this position in the structure of the Shb17 complex with FBP, which was annotated as a water molecule (Figure 3C), but potentially it might also represent a metal ion.…”

YjeQ, an Essential, Conserved, Uncharacterized Protein from Escherichia coli, Is an Unusual GTPase with Circularly Permuted G-Motifs and Marked Burst Kinetics

“…Values of k cat and K m were 8.1 h -1 and 0.41 mM for His-tagged YjeQ and 9.4 h -1 and 0.12 mM for untagged YjeQ, respectively. The steady state rate of GTP hydrolysis observed with YjeQ is comparable to those of p21H-Ras and E. coli EF-Tu, which have intrinsic GTPase turnover numbers of 1.7 and 2.2 h -1 , respectively ( , ).…”

“…Dialysis, for example, with 20 mM EDTA, followed by dialysis to remove EDTA and restore Mg 2+ to 10 mM in buffer abolished hydrolytic activity (data not shown). Bound GDP therefore appeared to be critical to the stability of purified YjeQ, a property common to many GTPases, including H-Ras (33) and EF-Tu (34). The nucleotide binding activity of the purified enzyme was a Nucleotide hydrolysis by pure YjeQ was assessed through measurement of the amount of inorganic phosphate produced using a malachite green/ammonium molybdate colorimetric assay (20).…”

Salmonella entericaSerovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

“…Our steady-state kinetic studies indicate that BipA alone hydrolyzes GTP at a low rate (k cat ϳ 22 h Ϫ1 ). The steady-state rates of GTP turnover by BipA is slightly higher than rates of p21H-Ras, E. coli EF-Tu, and E. coli YjeQ, which are 1.7, 2.2, and 8.1 h Ϫ1, respectively (14,17,45) but is lower than those reported for Thermotoga maritima and E. coli EngA (4,43). Upon the addition of 30S or 50S ribosomal subunits, a slight increase in k cat was observed, whereas a greater increase was seen in the presence of intact ribosomes.…”