The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+

Schweins, Thomas; Scheffzek, Klaus; Assheuer, Ralf; Wittinghofer, Alfred

doi:10.1006/jmbi.1996.0814

Cited by 59 publications

(44 citation statements)

References 57 publications

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“…On the other hand, the value of k cat for GTP hydrolysis in the presence of Mn 2ϩ is 0.069 min Ϫ1 , greater than that observed in the presence of Mg 2ϩ . The effect of Mn 2ϩ on accelerating hydrolysis of GTP over that observed with Mg 2ϩ has been observed also for the intrinsic GTPase activity of the ras proteins p21, Ran, and Rap1A (44). As seen in Table II, the kirromycin-stimulated GTPase activity in the absence of programmed ribosomes follows a similar dependence on added divalent metal ion to that observed for intrinsic GTPase activity.…”

Section: Role Of Mg 2ϩ In Ef-tu Actionsupporting

confidence: 57%

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

Rütthard

Banerjee

Makinen

2001

Journal of Biological Chemistry

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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...

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Section: Role Of Mg 2ϩ In Ef-tu Actionsupporting

confidence: 57%

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

Rütthard

Banerjee

Makinen

2001

Journal of Biological Chemistry

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“…For the interaction of Ras with its cognate RasGAP proteins, different kinetic mechanisms have been observed. For p120GAP, very fast association of substrates and release of products were found, and the chemical reaction is believed to be rate limiting (2,38,65). A completely different situation is observed during neurofibromin (NF1)-catalyzed hydrolysis of Ras-GTP, during which binding is also fast, but the overall reaction is limited by either the phosphate or Ras-GDP release step (5,38).…”

Section: Resultsmentioning

confidence: 99%

Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

Seewald

Kraemer

Farkašovský

et al. 2003

Molecular and Cellular Biology

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RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product P i . It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 ؋ 10 8 M ؊1 s ؊1 ). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.The GTP-binding protein Ran, which belongs to the superfamily of Ras-like guanine-nucleotide binding proteins, is both a key regulator of nuclear transport (22, 44) and a marker of chromosome position in spindle formation and nuclear envelope assembly (29) of eukaryotic cells. RCC1, the nucleotide exchange factor for Ran, localizes to histones H2A and H2B (52) and consequently creates a high concentration of Ran-GTP in the vicinity of the chromatin (11, 54). RanGAP, the GTPase-activating protein specific for Ran, increases the hydrolysis rate 10 5 -fold (35). During interphase, it is exclusively cytosolic (32) and, together with RCC1, creates a gradient of Ran-GTP across the nuclear membrane (34), which is the major driving force and determiner of directionality for nuclear transport (22,51). During mitosis, Ran-GTP induces spindle formation (75) and nuclear envelope assembly (78,79). For the latter, Ran-GTP hydrolysis accelerated by RanGAP is required (28), although it is not obvious how RanGAP is prevented from abolishing the Ran-GTP gradient. It is also not clear how the function of Ran in spindle and nuclear envelope formation can be applied to lower eukaryotes such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, which undergo a closed mitosis, whereas vertebrates go through the process of disassembly and reassembly of the nuclear membrane (open mitosis).The presence or absence of Ran-GTP in nucleosol or cytosol, respectively, is the determining factor for Ran-dependent cargo transport (22). Import receptors of the importin-␤ family bind to cargo in the absence of Ran-GTP and release it in the nucleus, where they bind with high affinity to Ran-GTP.In contrast, export cargo binding to export receptors (exportins) requires the presence of Ran-GTP. The dissociation of Ran-GTP complexes with importins and exportins on the cytoplasmic side of t...

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“…Examples in which metal ion identity affects the apparent pK a of proteincatalyzed reactions include the nucleotidyl transfer by a viral RNA polymerase and GTP hydrolysis by p21 ras (Schweins et al 1997;Bowers et al 2007;Castro et al 2007). Further testing of these relationships should be possible using enzymes in which (1) the cation does not play an obligatory role in the catalytic mechanism; (2) general acid-base catalysis is the ratelimiting step across the pH range examined; and (3) both pK a values can be experimentally measured.…”

Section: Smith Et Almentioning

confidence: 99%

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK_a

Smith

Mehdizadeh²,

Olive³

et al. 2008

RNA

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Several small ribozymes employ general acid-base catalysis as a mechanism to enhance site-specific RNA cleavage, even though the functional groups on the ribonucleoside building blocks of RNA have pK a values far removed from physiological pH. The rate of the cleavage reaction is strongly affected by the identity of the metal cation present in the reaction solution; however, the mechanism(s) by which different cations contribute to rate enhancement has not been determined. Using the Neurospora VS ribozyme, we provide evidence that different cations confer particular shifts in the apparent pK a values of the catalytic nucleobases, which in turn determines the fraction of RNA in the protonation state competent for general acid-base catalysis at a given pH, which determines the observed rate of the cleavage reaction. Despite large differences in observed rates of cleavage in different cations, mathematical models of general acid-base catalysis indicate that k 1 , the intrinsic rate of the bond-breaking step, is essentially constant irrespective of the identity of the cation(s) in the reaction solution. Thus, in contrast to models that invoke unique roles for metal ions in ribozyme chemical mechanisms, we find that most, and possibly all, of the ion-specific rate enhancement in the VS ribozyme can be explained solely by the effect of the ions on nucleobase pK a . The inference that k 1 is essentially constant suggests a resolution of the problem of kinetic ambiguity in favor of a model in which the lower pK a is that of the general acid and the higher pK a is that of the general base.

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The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+

Cited by 59 publications

References 57 publications

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

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

Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK_a

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The role of the metal ion in the p21ras catalysed GTP-hydrolysis: Mn2+ versus Mg2+

Cited by 59 publications

References 57 publications

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

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

Biochemical Characterization of the Ran-RanBP1-RanGAP System: Are RanBP Proteins and the Acidic Tail of RanGAP Required for the Ran-RanGAP GTPase Reaction?

The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pKa

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The ionic environment determines ribozyme cleavage rate by modulation of nucleobase pK_a