From the Laboratorium für Biochemie,
Universität Bayreuth, 95440 Bayreuth, Germany and the
§ Department of Biochemistry and Molecular Biology, Cummings
Life Science Center, The University of Chicago, Chicago, Illinois
60637
Received for publication, March 8, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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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 Mg2+-free protein has been investigated.
The intrinsic GTPase activity under single turnover conditions varied
according to the series: Mn2+ (0.069 min 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 In all G-proteins defined hitherto by x-ray crystallographic studies,
the substrate analog guanosine 5'-( 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 nucleotide-free elongation factor Tu
(17-20) and p21ras (7) has also allowed
comparison of divalent metal ions on nucleotide binding and hydrolysis
(8, 19, 21, 22) and the investigation of nucleotide-protein
interactions by magnetic resonance methods (16, 21-26). In these
studies, however, the question of whether the divalent metal ion is an
absolute requirement for GTP hydrolysis has not been addressed.
In the present study we report results of kinetic and equilibrium
binding studies to compare the influence of divalent metal ions on the
intrinsic and kirromycin-stimulated GTPase activity of EF-Tu of
T. thermophilus in the absence of programmed ribosomes. We
compared the influence of Mn2+ and VO2+ to that
of Mg2+ because they have been employed as paramagnetic
probes of Mg2+ function in thermostable EF-Tu (21, 23).
Because we have been able to show that EF-Tu when depleted of
nucleotide is not associated with protein-bound Mg2+, we
have been able to determine not only the influence of different divalent metal ions on the biological activity of EF-Tu without interference from adventitious Mg2+ but also the intrinsic
GTPase activity of the Mg2+-free protein. The results lead
to the conclusion that Mg2+ is not a catalytically
obligatory cofactor in the intrinsic and kirromycin-stimulated GTPase
activity in the absence of programmed ribosomes. We also show that the
binding of divalent metal ions provides the thermodynamic driving force
for stabilization of the EF-Tu·Me2+·GDP complex, the
effect being greatest with Mg2+. The differential role of
Mg2+ in EF-Tu function is discussed further through
analysis of thermodynamic relationships governing the hydrolysis of
nucleoside 5'-triphosphates (27, 28) and the kinetic requirement for a
nucleotide-exchange factor in the cell.
Materials
General--
Guanosine nucleotides and BSA were obtained from
Roche Molecular Biochemicals (Mannheim, Germany),
[8-3H]GDP and [8-3H]GTP from Amersham
Pharmacia Biotech, and [ Preparation of Nucleotide-free EF-Tu--
EF-Tu was obtained as
the EF-Tu·Mg2+·GDP complex from T. thermophilus (17) or from the cell paste of E. coli
(JM109) engineered with the tuf1 gene of T. thermophilus (HB8) (29). Nucleotide- and Mg2+-free
EF-Tu (EF-Tuf) was isolated as described by Limmer et
al. (16), as modified previously (21). For removal of GDP and Mg2+, EF-Tu was diluted 1:6 into 5 M urea
buffered to pH 5.65 with 0.01 M
(NH4)2HPO4. The resultant mixture
with a protein concentration of ~1.5 × 10 Methods
Atomic Emission Spectroscopy--
The Mg2+ content
of protein, nucleotide, kirromycin, and of all buffer solutions was
determined by inductively coupled plasma atomic emission spectroscopy
with a GBC Integra XMP spectrometer (BDC Scientific Equipment, Ltd.,
Dandenong, Victoria, Australia). The detection limit of this method is
1.0 × 10 Determination of Nucleotide Binding Affinity--
We observed
previously that variability in the stoichiometry of nucleotide binding
and biphasic character in Scatchard plots (30, 31) are essentially
eliminated when BSA is added to the incubation mixture (21). Because
nucleotide binding to BSA alone was not detected under these
conditions, we concluded that BSA provided the role of a protectant
neutral macromolecule, as is observed in a variety of biochemical
assays.2 For these reasons,
the use of BSA was continued in studies to determine nucleotide binding
affinity of EF-Tuf. The dissociation constants and the
stoichiometry of GDP and GTP binding to EF-Tuf were
determined following the nitrocellulose filter binding method of Arai
et al. (9, 12), as described in previous studies (21). For
binding affinity measurements, all stock solutions of protein, buffer,
and nucleotide were treated with Chelex to remove possible trace
contaminant divalent metal ions. The reaction mixture (0.0175 ml)
containing 0.05 M KCl, 8.8 × 10
To determine nucleotide binding affinity in the presence of
VO2+, low concentrations of the metal ion must be used to
avoid its precipitation as polymeric VO(OH)2 at neutral pH
(32). We found the optimal concentration of VO2+ to be
~1.0 × 10
After addition of the metal ion, the reaction mixture was incubated for
15 min at ambient temperature (~20 °C) according to Arai et
al. (9, 12), and aliquots of the reaction mixture for measurements
in triplicate were applied to the nitrocellulose membrane (HA 0.54 µm; Millipore Corporation, Belford, MA). The filter was then washed
thrice with 1 ml of an ice-cold solution of the reaction buffer
containing only 0.05 M KCl and 0.05 M HEPES at
pH 7.5. The protein-bound radioactivity retained by the filter was
measured after air drying with the aid of a Packard Instruments Minax Determination of GTPase Activity--
Intrinsic GTPase activity
of EF-Tuf was determined by adapting the method of Peter
et al. (36), as previously described (21). The reaction
mixture contained either 0.05 M KCl, 0.15 M
NH4Cl, and 1.0 × 10 Removal of Mg2+--
In a variety of studies, the
influence of divalent metal ions on the intrinsic,
kirromycin-stimulated, and physiological GTPase activity of EF-Tu has
been examined for EF-Tu of both E. coli (37, 38) and
thermophilic (19, 20, 24) organisms. Also, 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). In these studies, however, the
question of whether Mg2+ is a catalytically obligatory
cofactor for GTP hydrolysis could not be addressed directly because the
purified protein was associated with significant quantities of
nucleotide and Mg2+. An important motivation of the
investigations reported here was to differentiate the role of
Mg2+ in GTP hydrolysis from that in nucleotide binding and
to determine whether the divalent metal ion is an absolute requirement
for GTPase action. For this reason it was necessary to ensure efficient removal of protein-bound Mg2+.
We found that Mg2+ could be consistently removed with high
efficiency only under partial denaturation of EF-Tu in 5 M
urea at pH 5.6 followed by cation exchange chromatography. As shown in Fig. 1, the concentration of
Mg2+ bound as the EF-Tu·Mg2+·GDP complex
shows a linear dependence on protein concentration. However, the amount
of Mg2+ associated with EF-Tuf, when prepared
as described under "Experimental Procedures," was consistently
below the detection limit of the atomic emission instrumentation. From
Fig. 1 we can conclude that there must be less than 0.026 moles of
Mg2+ bound per mole of protein. By similar methods we
confirmed that the Mg2+ content of all other solutions,
including nucleotides and kirromycin, was also equivalently
negligible.
Wittinghofer and Leberman (18) proposed that Mg2+ is bound
to EF-Tu as the Mg2+-nucleotide complex. For this reason we
separated Mg2+ from the EF-Tu·Mg2+·GDP
complex together with the nucleotide by partial denaturation and cation
exchange chromatography. Extensive dialysis of the B. stearothermophilus EF-Tu·Mg2+·GDP complex against
nucleotide- and Mg2+-free buffer results in removal of only
55% of the nucleotide (18). In this respect we note that the
stoichiometry of protein-bound Mg2+ in Fig. 1 for the
EF-Tu·Mg2+·GDP complex is equivalent to about 0.6 moles
Mg2+ per mole of EF-Tu despite similar extensive dialysis
over a 24 h period. These correlative observations affirm the need
to remove nucleotide by denaturation followed by cation exchange
chromatography to achieve efficient depletion of protein-bound
Mg2+, as described under "Experimental Procedures."
Nucleotide Binding--
Whereas the emphasis of our studies was to
evaluate whether Mg2+ is a catalytically obligatory
cofactor in the intrinsic GTPase activity of EF-Tu, we first summarize
results of measurements of nucleotide binding affinity in the absence
and presence of divalent metal ions. In this study we compared the
influence of the divalent metal ions Mn2+ and
VO2+ to that of Mg2+. The former has been
employed in studies of EF-Tu of B. stearothermophilus as a
paramagnetic substitute for Mg2+ (8, 24) whereas the latter
paramagnetic species has been found to closely mimic structural
interactions of Mg2+ with nucleotides (33, 34).
The binding affinity of GTP and GDP to EF-Tuf in the
absence and presence of added divalent metal ions was evaluated on the basis of Scatchard plots (30, 31). Table
I summarizes all of our results on
nucleotide binding affinity of GDP and GTP to EF-Tuf in the
absence and presence of divalent metal ions. The Kd of 3.7 × 10
The influence of other divalent metal ions on nucleotide binding by
T. thermophilus EF-Tu has not been previously investigated. Whereas Mg2+ and Mn2+ show equivalent effects
in the binding of GDP to EF-Tu, as seen in Table I, the affinity for
GDP in the presence of VO2+ is decreased by a factor of
~5. On the other hand, both VO2+ and Mn2+
facilitate tighter binding of GTP than does Mg2+. It is of
interest to note that the binding affinity is not only decreased by one
to two orders of magnitude in the absence of added divalent metal ion
but also both nucleotides are bound with equal affinity.
GTPase Activity--
Fig. 2
illustrates progress curves for EF-Tuf-catalyzed hydrolysis
of GTP in the presence of Mg2+, Mn2+, and
VO2+ and in the absence of added divalent metal ion under
single turnover conditions. The activity in the presence of
Mn2+ is greater than in the presence of Mg2+,
which in turn is identical to the hydrolytic activity in the absence of
added divalent metal ion. The intrinsic GTPase activity of
EF-Tuf in the absence of divalent metal ion has not been
previously reported, and the magnitude of the effect is comparable with
that in the presence of Mg2+. For these reasons we took
special precautions to ensure that the protein and all reagents were
exhaustively depleted of adventitious Mg2+, as described
under "Experimental Procedures."
Whereas Scatchard analyses summarized in Table I showed that binding of
both GDP and GTP in the presence of VO2+ is of greater
affinity than in the absence of added divalent metal ion, we see in
Fig. 2 that hydrolysis of GTP in the presence of VO2+ is
slower than in the absence of divalent metal ions or in the presence of
Mg2+. Whereas we are not able to provide a complete
explanation of this difference at present, the affinity of
VO2+ for inorganic triphosphate to form a 1:1 complex is at
least 2 orders of magnitude greater than that of Mg2+ (40).
This effect is probably because of increased covalency in metal-ligand
interactions, which may hinder hydrolysis of the triphosphate moiety in
the protein-nucleotide complex. The apparent inhibitory effect of
VO2+ can be abolished by substitution of
NH4+ for K+ in the reaction mixture
whereas the reaction in the presence of Mg2+ or
Mn2+ is not influenced by addition of
NH4+. Because
VO2+ can be coordinated by amines and other nitrogen-donor
ligands3 (41-43), the high concentration of
NH4+ is likely to cause coordination to
VO2+, converting the system to be equivalent to that in the
absence of added divalent metal
ion.4
Table II compares the results of our
studies showing the influence of divalent metal ions under single
turnover conditions for both intrinsic GTPase and kirromycin-stimulated
GTPase activity in the absence of programmed ribosomes. For
measurements of intrinsic GTPase activity, the value of
kcat of 0.037 min
The hydrolysis of GTP catalyzed by EF-Tuf under multiple
turnover conditions is illustrated in Fig.
3. It is seen that substrate turnover is
faster in the presence of Mn2+ than in the presence of
Mg2+. On the other hand, substrate turnover is slower in
the presence of Mg2+ than in its absence. This latter
observation affirms that significant amounts of contaminant
Mg2+ could not have been present in the metal-free case
under multiple turnover conditions because substrate turnover is faster
in the absence of added divalent metal ion. Substrate hydrolysis under multiple turnover conditions occurs because of a combination of the
intrinsic GTPase action of the protein together with nucleotide exchange. The different pattern of the influence of metal ions under
multiple turnover conditions, therefore, must take into account the
relative catalytic effects observed under single turnover conditions,
as modulated by metal-dependent variations in the on- and
off-rate constants for substrate binding and product release.
As evident in Table I, the affinity of EF-Tuf for GDP is
identical in the presence of Mg2+ and Mn2+
whereas the affinity for GTP is slightly greater in the presence of
Mn2+. Therefore, because the rate of exchange of GDP by GTP
must be essentially the same in the presence of Mn2+ and
Mg2+, the enhanced rate of GTP hydrolysis with
Mn2+ may be because of either a decreased off-rate constant
for GTP binding under multiple turnover conditions or a different
involvement chemically of the metal ion in the reaction. Because the
kinetics of product release do not influence the single turnover
results, we attribute the enhanced intrinsic GTPase activity of the
Mn2+-enzyme to these same factors. Fig. 2 shows that the
intrinsic GTPase activity of the enzyme in the absence of added
divalent metal ion is unaltered from that in the presence of
Mg2+. The binding of GDP in the absence of divalent metal
ion is significantly weaker than in the presence of Mg2+ or
Mn2+, allowing even faster exchange of GDP by GTP under
multiple turnover conditions. Because the intrinsic GTPase activity in
the absence of added divalent metal ion is unaltered under single
turnover conditions, despite a lower affinity for GTP, the hydrolytic
activity in multiple turnover experiments must be considered as only
apparently enhanced over that of the Mg2+-enzyme because
of faster nucleotide exchange.
The GTPase activity of EF-Tu of T. thermophilus
depleted of bound nucleotide and divalent metal ion has not been
previously investigated in the absence of added divalent metal ions.
The main observation, as demonstrated through Figs. 2 and 3 and Table II, is that the intrinsic GTPase activity and the kirromycin-stimulated GTPase activity of T. thermophilus EF-Tu depleted of
protein-bound Mg2+ is equivalent to that in the presence of
added Mg2+. This result means that Mg2+ does
not have a direct catalytic role in the intrinsic and
kirromycin-stimulated hydrolysis of GTP catalyzed by EF-Tu in the
absence of programmed ribosomes. Polypeptide chain elongation on the
ribosome is estimated to proceed at a rate of ~600 peptide
bonds/ribosome/minute (45) requiring hydrolysis of one GTP per
elongation step. Because this is significantly greater than the value
of kcat of ~0.037 min The differential influence of divalent metal ions on GTPase activity
and the nucleotide binding affinity can be analyzed on the basis of
thermodynamic relationships in Scheme 1.
For the closed system of reversible reactions constructed in Scheme 1 for hydrolysis of the free Me2+·GTP complex and,
correspondingly, of the EF-Tu·Me2+·GTP complex, the
reactions for GTP and GDP binding and release run along the vertical
direction whereas the hydrolysis of free and EF-Tu-bound
Me2+·GTP are represented horizontally. The protein-bound
and free states of the Me2+·GTP and
Me2+·GDP complexes are coupled through equilibria for the
binding and release of each Me2+-nucleotide complex by
EF-Tu. The free energy change associated with binding of each metal
ion-nucleotide complex is estimated on the basis of the equilibrium
(dissociation) constants in Table I. As the best approximation of the
free energy change for hydrolysis of the free Me2+·GTP
complex, we apply the values determined through calorimetric and
titration studies of George and co-workers (27, 28) for adenosine
5'-triphosphate under conditions of pH, ionic strength, and
Mg2+ concentration corresponding to those in our
investigations. Because the closed series of linked equilibria in
Scheme 1 requires that 1) > Mg2+ (0.037 min
1) ~ 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 (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+ < Mn2+ ~ Mg2+ whereas the binding affinity of GTP 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 ~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.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-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 Mg2+ as the
naturally occurring cofactor in the cell. However, in EF-Tu1 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.
,
-imido)-triphosphate is bound as a complex with Mg2+ 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
Mg2+ 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 Mg2+ 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.
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
-32P]GTP from Hartmann
Analytik (Braunschweig, Germany). Vanadyl sulfate hydrate was obtained
from Aldrich (Milwaukee, WI) and Chelex 100 resin (dry mesh 50-100)
was obtained from BioRad Laboratories (Hercules, CA). HEPES was
purchased from Sigma (St. Louis, MO). All other reagents were of
analytical reagent grade. Kirromycin was a gift from Gist-Brocades
(Leiden, The Netherlands).
4
M was applied to a 1 × 52 cm column of CM-Sephadex
CL-6B (Sigma) and eluted with the same buffer to remove GDP. A second
buffer consisting of 0.01 M
(NH4)2HPO4 at pH 7.5, 5 M urea, and 0.5 M KCl was then applied to elute
the protein. Renaturation of the protein was carried out by dialyzing
against 0.05 M KCl and 0.15 M NH4Cl
or against 0.2 M KCl buffered with 0.05 M HEPES
at pH 7.5. An Amicon YM-10 membrane (Amicon, Inc., Beverly, MA)
extensively washed prior to use was employed to concentrate the protein
solution. Whereas the original description for chromatography of EF-Tu
and removal of Mg2+ includes 0.01 M EDTA in the
buffer (16), we found no difference in the results if EDTA was not
present during denaturation and cation exchange chromatography of the
protein. Therefore, to compare the influence of metal ions on
nucleotide binding and GTPase activity, EDTA was not used during
nucleotide removal to avoid the possible influence of low levels of
adventitious EDTA. Use of glass containers was also avoided to prevent
contamination with trace divalent metal ions. EF-Tuf
prepared in this manner was stored at
20 °C in the dialysis buffer
containing 50% (v/v) glycerol.
4 g Mg2+ per liter or 4.0 × 10
6 moles (g atoms) Mg2+ per liter of solvent.
9
M EF-Tuf, and 1.6 × 10
5 g
BSA/ml was buffered to pH 7.5 with 0.05 M HEPES. In the
reaction mixture the concentration of guanosine nucleotides varied from 12.5 × 10
9 to 500 × 10
9
M with [8-3H]GDP or [8-3H]GTP
of specific activity 1.5 × 10
6 Ci/nmol. For
nucleotide binding in the presence of Mg2+ or
Mn2+, metal ion was added to a final concentration of 0.01 M. Variation of the order of addition of Mg2+
or Mn2+ with respect to the addition of nucleotide was not
observed to influence binding affinity. However, this was not the case
for nucleotide binding in the presence of VO2+.
4 M, lower concentrations
resulting in greater variability of the results and higher
concentrations tending to caused precipitate formation. Because
VO2+ may form tight complexes with nucleotides of 1:1 and
1:2 metal/nucleotide stoichiometry (33, 34), we investigated the
influence of the order of addition of reagents to the incubation
mixture. For nucleotide binding in the presence of VO2+, it
was found that addition of VO2+ had to follow addition of
nucleotide to the reaction mixture containing EF-Tuf and
BSA to observe reproducibly maximum binding affinity and 1:1
stoichiometry of binding of GDP or GTP. In binding assays with
VO2+, BSA dialyzed against 0.05 M KCl
containing 0.05 M HEPES at pH 7.5 was presaturated with a
4-fold molar excess of VO2+ to account for the tight
binding of this metal ion by BSA (35). Also the microcentrifuge tubes
were pretreated with VO2+-BSA. Pretreatment of BSA with
the other metal ions was not found to be necessary.
Tri-Carb 4000 Series Liquid Scintillation Counter (Packard Instruments, Meriden, CT 06450).
5 M
EF-Tuf, buffered to pH 7.5 with 0.05 M HEPES or
0.2 M KCl and 1.0 × 10
5 M
EF-Tuf similarly buffered with HEPES. The latter mixture
was used to prevent coordination of the vanadyl ion by the ammonium ion.3 In single turnover
experiments, GTP was added so that EF-Tuf remained in
slight (~1.05:1.00) excess. For multiple turnover experiments, GTP
and divalent metal ion were both added to a final concentration of
1.0 × 10
4 M. To determine the
kirromycin-stimulated GTPase activity, the reaction mixture
additionally contained 5.0 × 10
5 M
kirromycin. In both single and multiple turnover experiments, the
reaction was initiated by addition of [
-32P]GTP of
specific activity 5.0 × 10
7 Ci/nmol. To quench the
reaction and to obtain maximum chromatographic resolution, aliquots of
the reaction mixture were removed with time and added 1:3 (v/v) to
ice-cold formic acid. The quenched aliquots were then stored on ice
until application for chromatography on polyethyleneimine-cellulose
sheets (Machery-Nagel, Dueren, Germany). Electronic autoradiography was
carried out as described (21). The data were fitted to a single
exponential curve with fixed baseline offset by a nonlinear
least-squares method with the aid of the program GRAFIT3.0 (Erithacus
Software, Ltd., Staines, UK).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
View larger version (14K):
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Fig. 1.
Mg2+ content of EF-Tu determined
by inductively coupled plasma atomic emission spectroscopy.
Untreated EF-Tu·Mg2+·GDP as purified from the cell
( ); Mg2+- and nucleotide-free EF-Tu (
).
9
M for the binding of GDP in the presence of
Mg2+ is in good agreement with estimates of 1.8 × 10
9 M and 1.1 × 10
9
M made by Wagner et al. (10) and Arai et
al. (9, 12), respectively, for the T. thermophilus
protein. Furthermore, Wagner et al. (10) report a
Kd of 29 × 10
9 M
for the binding of GTP in the presence of Mg2+ that was
obtained by kinetic stopped-flow measurements of ligand displacement
using protein tryptophan fluorescence. Our result based on a
nitrocellulose binding assay of [8-3H]GTP is in good
agreement with the stopped-flow measurement of Wagner et al
(10). On this basis we can conclude that very little GTP hydrolysis
occurred under the experimental conditions for Scatchard analysis.
Comparison of stoichiometric binding coefficients and dissociation
(equilibrium) binding constants of GDP and GTP to EF-Tuf in the
presence or absence of added divalent metal ions determined by
Scatchard plot analyses
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Fig. 2.
Hydrolysis of EF-Tu-bound GTP determined by
release of [32P]phosphate from
[ -32P]GTP catalyzed by
EF-Tuf under single turnover conditions. The protein
was added to the reaction mixture (0.05 M KCl buffered to
pH 7.5 with 0.05 M HEPES) to a concentration of 1.0 × 10
5 M. The reaction in this case was
monitored for incubation at 37 °C. The added metal ions
Mn2+ (
) and Mg2+ (
) were added to 0.01 M concentration, whereas VO2+ (
) was added
to a final concentration of 1.0 × 10
5
M. (
indicates no added divalent metal ion.) Values of
kcat, obtained by non-linear least-squares fit
of the data to a single exponential function, are reported in Table
II.
1 in the presence
of Mg2+ is in good agreement with that of 0.042 min
1 reported by Zeidler et al. (11). On the
other hand, the value of kcat for GTP hydrolysis
in the presence of Mn2+ is 0.069 min
1,
greater than that observed in the presence of Mg2+. The
effect of Mn2+ on accelerating hydrolysis of GTP over that
observed with Mg2+ 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.5
Comparison of kinetic rate constants for intrinsic and
kirromycin-stimulated GTPase activity of EF-Tuf under single
turnover conditions in the presence or absence of added divalent metal
ion
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Fig. 3.
Comparison of the intrinsic GTPase activity
of EF-Tu under multiple turnover conditions. The reaction mixture
is as described under "Experimental Procedures." GTPase activity in
the presence of 1 × 10 4 M
Mn2+ (
); 1 × 10
4 M
Mg2+ (
); and in the absence of added metal ion (
).
The GTP/ EF-Tu ratio was 10:1 with a protein concentration of 1.0 × 10
5 M.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 observed
for intrinsic GTPase activity, as reported here and by others (11, 46),
the rate-limiting step cannot be the same as for the physiological
reaction of EF-Tu-catalyzed GTP hydrolysis in the presence of
programmed ribosomes. If the change in rate-limiting step in the
presence of programmed ribosomes is a consequence of direct
participation of Mg2+ in catalysis, structural
relationships of the divalent metal ion and the triphosphate moiety of
GTP in the active site of the EF-Tu·Mg2+·GTP complex
must differ significantly from those described heretofore (5, 6). Thus,
the results of our studies indicate not only that our understanding of
the structural and chemical role of Mg2+ in the biological
function of EF-Tu is incomplete but also that the physiological
relevance of the x-ray structure of the
EF-Tu·Mg2+·guanosine 5'-(
,
-imido)-triphosphate
complex (5, 6), prepared and crystallized in the absence of
ribosomes through methods comparable with those applied here, is unclear.
Gi = 0, the influence of divalent
metal ions on the EF-Tu-catalyzed hydrolysis of the
Me2+·GTP complex with respect to the corresponding
hydrolytic reaction of the free Me2+·GTP complex in
solution can be quantified. This analysis assumes that the free energy
change for hydrolysis of each Me2+·GTP complex in its
free form can be approximated by that for Mg2+·GTP.
Whereas the analysis is rigorously applied in the case of Mg2+, we anticipate that the error attributed to this
assumption for the other divalent metal ions, particularly
Mn2+, is not significant.
View larger version (11K):
[in a new window]
Scheme 1.
With respect to the G°' of
34.9 kJ/mol for hydrolysis of free
Mg2+·GTP in solution, the results in Table
III show that the differential binding
affinity of GDP and GTP by EF-Tuf in the presence of
divalent metal ions results in an increasing thermodynamic driving
force to stabilize the EF-Tu·Me2+·GDP complex according
to the series VO2+ < Mn2+ < Mg2+.
Whereas Mg2+ retards hydrolysis of GTP in the absence of
protein, a process that underlies the stability of nucleoside
5'-triphosphates in the cell, Mg2+ favors formation of the
EF-Tu·Mg2+·GDP complex by ~10 kJ/mol over the free
Mg2+·GDP complex under comparable conditions of ionic
strength, pH, and Mg2+ concentration. Furthermore, it is
clear that the protein alone is not responsible for this action because
EF-Tuf is not associated with differential binding affinity
for GTP and GDP in the absence of divalent metal ion. Also, the
thermodynamic driving force of Mg2+ to stabilize the
EF-Tu·Mg2+·GDP complex is independent of the catalytic
role that Mg2+ may have in the physiological GTPase
activity of EF-Tu in the presence of programmed ribosomes.
|
The analysis through Table III and Scheme 1 affirms the generally held
notion that the role of Mg2+ is to stabilize the
EF-Tu·Mg2+·GDP complex. This protein-nucleotide complex
in turn serves as a pivotal point in the regulation of the activity
cycle of EF-Tu. Because the cellular content of GTP is at least 7-fold
greater than that of GDP (47), EF-Tu-bound GTP upon hydrolysis in the cell would lead to saturation of the protein as the tightly bound EF-Tu·Mg2+·GDP complex simply because of the ubiquitous
presence of Mg2+. Thus, the thermodynamic role of
Mg2+ in EF-Tu catalyzed GTP hydrolysis requires the
presence of a nucleotide exchange factor in the cell to kinetically
facilitate replacement of GDP by GTP. Comparison of the x-ray
structures of the GTP (5, 6) and GDP (48, 49) bound forms of EF-Tu shows significant structural changes involving not only local changes
in the metal ion and nucleotide binding sites but also global changes
involving shifts of domains II and III. The binding surfaces on EF-Tu
involved in binding to EF-Ts are located in the G domain and domain
III, and their arrangement remains similar upon formation of the
EF-Tu·EF-Ts complex to that in the EF-Tu·Mg2+·GDP
complex. It is thought that this structural relationship underlies the
kinetic preference of EF-Ts to bind to the GDP complex of EF-Tu over
the GTP complex (50). On this basis, it is likely that the role of
Mg2+ in stabilizing the GDP complex facilitates recognition
of the EF-Tu·Mg2+·GDP complex by EF-Ts. It is of
interest to note that binding of T. thermophilus EF-Tu by
the nucleotide exchange factor EF-Ts results in insertion of the Phe-82
residue of EF-Ts into the hydrophobic pocket of EF-Tu formed by His-85,
Leu-122, and His-119, disturbing the Mg2+-phosphate
portion of the active site (50), and the occupancy of Mg2+
in the binding pocket, as established by x-ray studies of the Tu·Ts
complex (50, 51), is abolished. Thus, in the biological action of
EF-Tu, Mg2+ binding stabilizes the conformation of the
EF-Tu·Mg2+·GDP complex to facilitate recognition by
EF-Ts, but destabilization of Mg2+ binding in the active
site of Tu is likely to be critical to initiate nucleotide exchange.
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ACKNOWLEDGEMENT |
---|
We thank Professor Mathias Sprinzl for helpful discussions and support.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants MCB-9513538 and MCB-0092524 from the National Science Foundation, Grant CRG960629 from NATO, and Grant Sp 243/-2 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry & Molecular Biology, The University of Chicago, 920 East 58th St., Chicago, IL 60637. E-mail: makinen@uchicago.edu.
Published, JBC Papers in Press, March 26, 2001, DOI 10.1074/jbc.M102122200
2 Proteins are known to rapidly denature when used in dilute solutions and therefore, many enzyme preparations are provided in a mixture with added BSA as a source of neutral protein, e.g., Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning, A Laboratory Manual (Nolan, C., ed) pp. 5.1-5.33, Cold Spring, Harbor Laboratory Press, NY.
3 We have confirmed by electron paramagnetic resonance experiments that VO2+ is coordinated by NH3 in the presence of NH4Cl under the concentration conditions employed in these studies (A. Banerjee and M. W. Makinen, unpublished observations).
4 We have observed by Scatchard analyses that the binding of nucleotide by EF-Tuf in the presence of VO2+ and 0.15 M NH4Cl results in values of dissociation constants equivalent to those observed in the absence of added divalent metal ion (A. Banerjee and M. W. Makinen, unpublished observations).
5 Because it was likely that VO2+ would have additional binding interactions with kirromycin, different from those of the other divalent metal ions studied, and because we had limited quantities of the antibiotic, we did not evaluate kirromycin-stimulated GTPase activity in the presence of VO2+.
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ABBREVIATIONS |
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The abbreviations used are: EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; EF-Tuf, nucleotide- and magnesium-free EF-Tu; Me2+, designation of a generic divalent metal cation; BSA, bovine serum albumin.
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