From the Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461-1926
Received for publication, October 11, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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The interdependence of GTP hydrolysis and
the second messenger functions of virtually all GTPases has stimulated
intensive study of the chemical mechanism of the hydrolysis. Despite
numerous mutagenesis studies, the presumed general base, whose role is to activate hydrolysis by abstracting a proton from the nucleophilic water, has not been identified. Recent theoretical and experimental work suggest that the The cellular activities of the Ras protein are causally linked to
tumorogenesis in a high percentage and wide variety of human tissues
(1). Binding of this cellular messenger to Raf-1 protein kinase
initiates a metabolic cascade that leads to cell growth and
differentiation (2, 3). Ras plays essential roles in regulating the
cell cycle (4, 5), apoptosis (6), mitogen-activated protein
kinase pathways (7, 8), and retroviral activation (9). The allosteric
interactions that mediate signaling between GTPases and their targets
are typically switched from on to off by the hydrolysis of GTP (10,
11); however, there are exceptions (12). The interdependence of
hydrolysis and signaling functions of GTPases has fostered intensive
studies of the chemical mechanism of the Ras-catalyzed hydrolysis
of GTP.
Classical general base theory suggests that Ras might catalyze
hydrolysis by abstracting a proton from the nucleophilic water, thereby
activating it to attack the The idea that the The materials and suppliers used in this study are as follows:
restriction enzymes (New England Biolabs), Pfu polymerase
(Stratagene), polyethyleneimine F-cellulose TLC plates (EM
Science), KCNO (Acros Organics), radionucleotides (PerkinElmer Life
Sciences), 18O-water (96.2 atom % 18O)
(Isotec), Q Sepharose Fast Flow resin (Amersham Pharmacia
Biotech), P6 spin columns (Bio-Rad), DNA primers
(Oligonucleotide Synthesis Facility, Albert Einstein College of
Medicine), BL21(DE3) Escherichia coli and pET-23a(+) plasmid
(Novagen), and Centricon 10 (Amicon). Buffers, media, enzymes,
nucleotides, PCl5, and solvents were the highest grades
available from Aldrich, Sigma, or Fisher.
Construction of the ras Expression
Vector--
Oligonucleotide-directed polymerase chain reaction was
used to amplify the DNA coding sequence for amino acids 1-166 of human Ras from a yeast expression plasmid using the following
oligonucleotides: 5'-ATTCCATATGACCGAATACAAACTGG-3' and
5'-GCGGATCCGTTAGTGCTGACGGATTTCACGAAC-3'. The amplified product
contained NdeI and BamHI restriction sites at the
3' and 5' ends, respectively, that were used to insert the
ras coding region into pET-23a(+). Each strand of the
ras coding region in the pET-23a(+)/ras
expression vector was sequenced. The primary sequence predicted by the
coding region exactly matched that of the published human
ras sequence (GenBankTM accession number
J00277). The expression system produced ras at The Ras Purification--
Ras was purified using standard
procedures (21, 22). Approximately 10 mg of >95% pure protein Ras was
obtained per liter of cell culture. The pure protein was stored in
buffer containing 0.10 mM GDP at The Ras Activity Assay--
GDP was removed from Ras stocks by
incubating Ras (52 µM, final concentration) for 5 min in exchange buffer (23, 24) (50 mM Tris (pH 7.5), 20 mM EDTA, 200 mM
(NH4)2SO4, 10 mM
dithiothreitol; T = 25 °C). The enzyme/exchange
solution was then loaded onto a P6 spin column (equilibrated in 50 mM Tris, pH 7.5, 10 mM dithiothreitol) and
eluted by centrifugation directly into 1.2 active site equivalents of
GTP/[ The Synthesis of
[
The nucleotide was purified on a Q Sepharose Fast Flow anion exchange
column using a 10 mM to 1.2 M triethylamine
bicarbonate (pH 7.6) linear gradient. GTP eluted at 1.0 M
triethylamine bicarbonate. Fractions containing GTP were collected and
rerun under similar conditions to further remove nucleotide
contaminants. Triethylamine was removed by rotary evaporation to
dryness; the GTP was suspended and rotary evaporation three
times in methanol and three times in water. The nucleotide was then
dissolved in water, and the pH was adjusted to 8.5 by addition of 10 M KOH. The purified product was stored at Electrospray Mass Spectrometry--
The nucleotides were
analyzed by mass spectrometry at the Hunter College Mass Spectrometry
Facility, New York, NY, on an Agilent Technologies (formerly Hewlett
Packard) HP1100 liquid chromatography/mass selective detector
instrument. Negative ion electrospray spectra were analyzed to give
89.6 and 88.0% enrichment in 18O for
[ Nucleotide Exchange--
Ras·GDP storage buffer was replaced
with nucleotide exchange buffer (200 mM
Tris-HCl, 200 mM
(NH4)2SO4, 0.55 mM
dithiothreitol, 0.5 mM NaN3, 0.6 mM
EDTA, pH 7.5) by repeated concentration and dilution at 4 °C using
Centricon 10 concentrators. After buffer exchange, Ras was adjusted to
~0.50 mM, and a 10-fold molar excess of
[ Sample Preparation for Sample Preparation for Amide I Experiments--
Ras·GDP was
exchanged into deuterized buffer (20 mM Tris-HCl, 10 mM MgCl2, 0.5 mM NaN3,
D2O solvent, pD*2
7.5). The pD was adjusted with DCl or NaOD. The final Ras concentration was 0.4 mM. Ras·GTP was exchanged into 20 mM
Tris-HCl, 0.5 mM NaN3, 0.6 mM EDTA,
D2O solvent, pD 7.5. The EDTA sequesters Mg2+
and prevents GTP hydrolysis. Concentrated protein was then added to
different buffers (chloroacetic acid, formic acid, and acetic acid)
containing MgCl2 just before the IR experiments.
Spectroscopy--
The IR studies of the phosphate stretch modes
involve isotope editing procedures that have been discussed
previously.1 All Fourier transform IR spectra were measured
on either an IFS-66 Fourier transform spectrometer (Bruker Instruments
Inc., Billerica, MA) or a Magna 760 Fourier transform spectrometer
(Nicolet Instrument Corp., WI). A dual cell shuttle accessory
was used to perform the 18O-16O difference
spectroscopy. This procedure makes sure the environments of two
subtracted samples are essentially identical. The Teflon spacer between
two BaF2 cells was set at 25 µm. Typically, the sample
and reference spectra are not exactly identical. In this case, the
parent spectra are factored such that the amide II bands are nulled
after subtraction. The protocol for buffer subtraction is identical
except that the baseline between 1750 cm Amide I Measurements--
The IR spectra of the protein
complexes in the amide I region were obtained at various pH values. The
spectra were enhanced by Fourier spectral deconvolution (FSD)
(enhancement factor = 1.5; intrinsic bandwidth = 17 cm The validity of the The vibrational spectrum of phosphate provides a sensitive,
site-specific measure of its protonation state. The frequencies of the
symmetric and antisymmetric modes of the P-O bonds of phosphate and
phosphate monoesters undergo large (~75
cm The vibrational spectrum of the Ras·GTP·Mg2+ complex is
quite complex, and identifying the stretch band of To aid in assigning the Ras·GTP·Mg2+ difference spectra
and to provide comparisons between the active site and solution
behavior of the -phosphate of GTP could be the general base.
The current study investigates this possibility by studying the pH
dependence of the vibrational spectrum of the
Ras·GTP·Mg2+ and Ras·GDP·Mg2+
complexes. Isotope-edited IR studies of the
Ras·GTP·Mg2+ complex show that GTP remains bound to Ras
at pH as low as 2.0 and that the
-phosphate is not protonated at
pH
3.3, indicating that the active site decreases the
-phosphate pKa by at least 1.1 pKa units compared with solution. Amide I studies
show that the Ras·GTP·Mg2+ and
Ras·GDP·Mg2+ complexes partially unfold in what appear
to be two transitions. The first occurs in the pH range 5.4-2.6 and is
readily reversible. Differences in the pH-unfolding midpoints for the
Ras·GTP·Mg2+ and Ras·GDP·Mg2+ complexes
(3.7 and 4.8, respectively) reveal that the enzyme-
-phosphoryl interactions stabilize the structure. The second transition, pH 2.6-1.7, is not readily reversed. The pH-dependent
unfolding of the Ras·GTP·Mg2+ complex provides an
alternative interpretation of the data that had been used to support
the
-phosphate mechanism, thereby raising the issue of whether this
mechanism is operative in GTPase-catalyzed GTP hydrolysis reactions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-phosphate. Careful inspection of the
GTP-bound active site of Ras suggests five possible general base
candidates (Asp-38, Asp-57, Gln-61, Glu-62, and glu-63). The GTPase
activity of mutants of each of these amino acids has been studied
(13-16), and it is generally agreed that the presumed base has not
been identified. The lack of a conclusive identification of the base
has spawned alternative suggestions for the chemical mechanism. A
recent theory that has attracted considerable attention is the
-phosphate general base theory, which hypothesizes that the
-phosphate of GTP, rather than an amino acid R-group, is the general base.
-phosphate might be the general base in
GTPase-catalyzed hydrolysis reactions was described first in a computational study that calculated that the hydrolysis
G
for this mechanism is 24 ± 5 kcal/mol, which is close to the experimentally observed value
(~22 kcal/mol) (17). Support for this theory was provided in a
subsequent paper in which the 31P-NMR of Ras-bound GTP was
studied as a function of pH. The
-,
-, and
-resonances of GTP
shift concomitantly at a pH midpoint of 2.9, which agrees, within
error, with the midpoint obtained from pH rate studies of Ras-catalyzed
GTP hydrolysis. pH rate studies of Ras kcat
mutants were used to construct a free energy plot
(
G
versus
pKa); the plot was linear, with a slope of 2.1 (18).
These observations are consistent with, but do not prove that, the
observed midpoint is the pKa of the
-phosphate of
the bound GTP. Vibrational spectroscopy provides an unambiguous means
of assigning the protonation state of phosphate and can be used to
define the structural and bonding changes that occur upon protonation
(20).1 To definitively assess
whether the midpoint is, in fact, the pKa of the
-phosphate and to define the bonding changes associated with the
protonation, the pH dependence of the vibrational spectrum of the
Ras·GTP·Mg2+ complex was investigated.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
10% of the
soluble cellular fraction (SDS-polyacrylamide gel electrophoresis).
70 °C.
-32P]GTP tracer. Ras was nucleotide-free for less
than 1 min. The GTP hydrolysis reaction was initiated by the addition
of MgCl2 (10 mM, final concentration)
and quenched by the addition of Na4EDTA, pH 9.5 (200 mM, final concentration). 10-min time points were taken,
spotted on polyethyleneimine F-cellulose TLC plates, and developed in a
0.90 M LiCl mobile phase. The radioactive reactants were
quantitated using an AMBIS two-dimensional radioactivity scanner. The
resulting time course fit a single exponential to give a calculated
turnover number of 0.013 ± 0.005 min
1.
-18O3]GTP--
460 µmol of fresh
PCl5 was added quickly to a 30-fold molar excess (250 µl)
of rapidly stirred 18O-water. The solution was brought to
pH 7.0 by addition of 10 M KOH. 460 µmol of sodium
acetate was then added from a 1.0 M stock. The mixture was
diluted 2-fold with unlabeled water, and 7.5 molar equivalents of KCNO
were added from a freshly prepared 6 M stock. The reaction
solution was maintained at 30 °C and kept at pH 6.5 by the addition
of glacial acetic acid in 5-min intervals over 30 min. Two additional
molar equivalents of KCNO were added, and pH monitoring was continued
for an additional 30 min. 4.0 ml of a solution containing ADP (115 mM (460 µmol)), MgSO4 (116 mM),
and Tris (0.58 M, pH 7.3) was added, followed by 17 units (µmol of substrate converted to product per min at
Vmax) of carbamate kinase, and the mixture was
incubated at 38 °C for 30 min to convert the ADP and carbamyl
phosphate to [
-18O3]ATP. The nucleotide
was diluted to 5.5 mM using an 11 mM Tris (pH
7.5), 11 mM MgSO4 buffer. Five mole equivalents
of GDP and 200 units of nucleoside-5'-diphosphate kinase were added,
and the mixture was incubated at 37 °C for 1 h.
70 °C. TLC
on polyethyleneimine F-cellulose plates showed that the
[
-18O3]GTP was more than than 95% pure.
-18O3]GTP.
-18O3]GTP was added. Nucleotide exchange
was complete after 30 min at 25 ± 2 °C.
-Phosphate Experiments--
Following
nucleotide exchange, nucleotide exchange buffer and excess nucleotide
were removed, as described above, and replaced by 20 mM
Tris-HCl, 10 mM MgCl2, 0.5 mM
NaN3, pH 7.5. The pH was adjusted by addition of HCl or
NaOH. The final concentration of Ras used in the experiments was 2 to
4 mM.
1 and
2000 cm
1 is nulled. The contribution from
vapor was also removed by subtraction.
1). The FSD method helps to resolve the
peak position of each component in the original spectra. From the FSD
spectra, we could determine the frequencies that characterize the high
and low pH forms. To find the pKa of the transition
from low to high pH, the difference in the intensity pairs that changed
the most during the titration were plotted as a function of pH
(1640-1635 cm
1 and 1641-1638
cm
1 for the Ras·GTP·Mg2+ and
Ras·GDP·Mg2+ complexes, respectively). Because of
slightly varying protein concentrations at the different pHs, the
intensity differences were normalized by the area of the corresponding
amide I band. A linear transformation of the data was performed to make
the differences lie between 0 and 1, and the resulting normalized intensity versus pH plots were fit using the
Henderson-Hasselbalch equation to obtain apparent
pKa values (see Fig. 3).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-phosphate general base hypothesis rests on
the proposition that the
-phosphate of Ras-bound GTP partially abstracts a proton from the nucleophilic water, making it more hydroxide-like, thereby activating it to attack the
-phosphate and
cleave the
,
-bond of GTP. Experimental support for this hypothesis is provided by 31P-NMR pH titration studies of
Ras·GTP·Mg2+ in which the
,
, and
-31P resonance positions shifted concomitantly at a pH
midpoint of 2.9. This midpoint was interpreted as the
pKa of the GTP
-phosphate in the Ras·GTP
complex (16). In solution, the
,
, and
-31P
resonances shift independently as they become protonated (25). If the
results of the enzyme-bound studies are interpreted on the basis of the
addition of a single proton to the tripolyphosphate chain of GTP, the
simultaneous shift of all three resonances creates an unavoidable
ambiguity regarding which of the three phosphoryl groups receives the
proton; it is as if the proton "belongs" to each of them.
1) shifts as phosphate switches between its
mono- and dianionic forms (20). In favorable cases (i.e.
when both the symmetric and antisymmetric frequencies of the oscillator
can be assigned) it is possible to quantitate changes in bond length
and bond order with accuracies of better than ±0.004 Å and 0.04 valence units,1 respectively. To obtain a more complete
description of the structural and bonding changes that the
-phosphate undergoes as the pH passes through the 2.9-midpoint, the
pH dependence of the vibrational spectrum of the
-phosphate of GTP
in the Ras·GTP·Mg2+ complex was studied using IR spectroscopy.
-phosphate of GTP is difficult (26). This problem can be overcome using an
isotope-editing scheme in which the natural abundance spectrum of
Ras·GTP·Mg2+ is subtracted from that of an identical
complex in which the nonbridging oxygen atoms of the GTP
-phosphate
have been enriched in 18O. Increasing the mass of an oxygen
nucleus shifts the vibrational frequencies of the modes in which it
participates; this frequency offset is what is revealed in the
difference spectrum. Properly subtracted parent spectra produce a
difference spectrum that contains only the differences in the
absorption bands associated with the bonds that involve the labeled
nuclei (26).1
-phosphate, the difference spectra for the
-phosphate-protonated and -deprotonated forms of
GTP·Mg2+ in solution were obtained. To select the pH
values at which to acquire the protonated and deprotonated spectra, the
pKa of the GTP·Mg2+ complex (4.2 ± 0.2) was determined in water at 25 °C (data not shown). The
unprotonated (pH 5.9), intermediate (pH 4.5), and fully protonated (pH
2.8) difference spectra are presented in Fig.
1A. The protonated and
deprotonated spectra do not change at higher and lower pH values,
respectively. The intermediate spectrum, pH 4.5, appears to be a
weighted composite of the protonated and deprotonated spectra,
suggesting that the system is well described by a two-state
(protonated/deprotonated) model.
View larger version (18K):
[in a new window]
Fig. 1.
pH dependence of the GTP
-Phosphate IR Difference Spectra. A, the
GTP·Mg2+ solution spectra. The sample composition was as
follows: [
-18O3]GTP or GTP (4.0 mM), MgCl2 (10 mM);
T = 20 ± 1 °C. B, the
Ras·GTP·Mg2+ spectra. The solutions contained the
following:
Ras·[
-18O3]GTP·Mg2+ or
Ras·GTP·Mg2+ (2.0 mM), Mg2+ (10 mM), Tris/Cl (20 mM), pH 7.5; T = 4 ± 1 °C. Samples were prepared and measurements were made
as described under "Materials and Methods."
The pH dependence of the Ras·GTP·Mg2+ difference
spectra is shown in Fig. 1B. The position and
18O-isotopic shift of the Ras-bound (pH 7.5) and solution
phase (pH 5.9) -phosphate difference spectra of
GTP·Mg2+ are extremely similar. The ~12
cm
1 spectral shift that occurs when the
nucleotide binds to Ras results from specific interactions between the
-phosphate and the active site (27, 28).1 These
similarities strongly support the notion that the
-phosphate is dianionic at the active site. The spectrum begins to change at pH 3.3, indicating that the
-phosphate remains largely
unprotonated at this pH. Thus, the active site of Ras has decreased the
pKa of the
-phosphate by >1.1 units compared
with solution. As the pH decreases below 3.3, the difference spectrum
becomes distinctly different, indicating a considerable change in the
electronic structure of the
-phosphate. However, the spectrum does
not resemble that for the protonated GTP·Mg2+ in
solution. Therefore, the spectral change below pH 3.3 cannot be
explained simply on the basis of a protonation of the
-phosphate. Whether or not
-phosphate protonation occurs, there must be a substantial change at the active site to yield the very unusual observed bands.
The conclusions from the IR data agree in many respects with previous
NMR results. The decreased pKa and change in the
-phosphate environment are consistent with the NMR results, as is
the fact that most (greater than 90%) of the nucleotide remains bound
at the low pH; if this were not the case, the protonated solution
spectrum would be observed. However, despite our efforts to reverse the
low pH transition, we could only partially recover the high pH
spectrum, suggesting that an irreversible event occurs at low pH. Given
the low pH of the event, it was probably caused by an unfolding
of the complex. To further investigate the unfolding issue, the pH
dependence of the amide I spectrum of the nucleotide-bound complex was studied.
The pH-dependent Unfolding of Ras--
Protein amide I
spectra (~1600-1700 cm1) arise from the
C=O stretch of the polypeptide backbone. The position of a C=O band within the amide I region is determined primarily by its hydrogen bonding and transition dipole coupling to other C=O groups, which is
distance- and orientation-dependent (29). The unique
geometric and bonding characteristics of the different secondary
structures result in structure-specific amide I absorbance bands.
Previous correlations between band position and secondary structure
(30, 31) have assigned absorbance centered at 1650-1655
cm
1 to the
-helix and absorbance at
1635 and 1675 cm
1 to the low (intense) and
high (weak) frequency components of the
-sheet structure; peaks at
1668, 1675, and 1686 cm
1 are indicative of
turns, and the 1645 cm
1 region is associated
with a disordered polypeptide. Despite ongoing efforts, accurate
quantitation of the secondary structural composition from amide I
spectra is not yet generally feasible (32); however, changes in an
amide I spectrum are frequently used as a sensitive indicator of
secondary structural change (33-36).
The amide I band of the Ras·GTP·Mg2+ and
Ras·GDP·Mg2+ complexes at three different pD values are
shown in Fig. 2, A and
B. The samples were prepared in D2O to shift the
intense IR absorption band of water out of the amide I region. To
better visualize the amide I regions that change as a function of pD,
the spectra were enhanced by FSD (37, 38). The FSD spectra are shown in
Fig. 2, C and D. The pD-dependent
changes in the amide I spectra of these complexes clearly indicate
changes in their secondary structure. The GTP complex undergoes two
denaturing transitions. The first, between pD* 5.4 and 2.6, is well
behaved and reversible. The second, between pD* 2.6 and 1.7, is not
well behaved. The results were not reproducible at pD* values below
2.7. We suspect that this is due to a tendency for Ras to aggregate at
this low pD* value, a common finding for acid-unfolded proteins.
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Ras contains large sections of -sheet and
-helix, and, as
expected, the amide I region of the FSD spectrum of the Ras-nucleotide complexes near neutrality show appropriate amplitude in the
-helical (1656 cm
1) and
-sheet (1635 and 1674 cm
1) regions. Low pH denatures both the
-helical and
-sheet structures, because the 1635 and 1651 cm
1 peaks in the high pD* are replaced by
peaks at 1640 and 1657 cm
1. At lower values
of pD, below 2.6, the changes in the amide I absorbance indicate
disruption of still more secondary structure in the second transition.
To better understand the pD dependence of the first (reversible)
transition, titration curves of the amide I changes were constructed
(Fig. 3). The curves were fit using the
Henderson-Hasselbalch equation (the fits are shown as solid curves).
The Ras·GDP·Mg2+ titration is fit well by a model in
which the observed changes are due to the addition of a single proton,
with a pKa of 4.8. The single-proton scheme did not
describe the Ras·GTP·Mg2+ titration well; however, a
two-proton uptake model provided an excellent fit. The apparent
pKa predicted by the fit was 3.7. The 1.1 pKa difference in the midpoints of the denaturation
curves for the GTP and GDP complex indicates that -phosphate·Ras
contacts stabilize protein toward acid-driven unfolding. The structure
of the protein is altered by these contact(s) such that the
unfolding appears to be simultaneous with the addition of approximately
two protons, instead of one. The apparent identity of the
pKa values of the groups protonated in the
transition can be explained either on the basis of synergism in the
binding of these protons or, more simply by, say, two carboxylates with pKa values within ~0.1 pKa
units of one another. Ligand-induced stabilization of the folded state
of proteins is well documented (39-41), and recent work has shown that
GDP and GDP·Mg2+ stabilize the urea-driven
unfolding of Ras by
5.7 and
12.3 kcal/mol, respectively (42,
43).
|
Conclusions--
The difference spectrum for the deprotonated GTP
-phosphate in solution resembles that for the
Ras·GTP·Mg2+ complex at pH
3.3 and differs
markedly from the protonated
-phosphate spectrum. It is clear from
this and other vibrational studies (26-28) that the GTP
-phosphate
is not protonated in the Ras·GTP·Mg2+ complex. The pH
dependence of the Ras·GTP·Mg2+ difference spectrum
indicates that the pKa of the
-phosphate of bound
GTP is
3.3, which is substantially lower than that for
GTP·Mg2+ in solution (4.2). Below pH 3.3, GTP remains
bound to the enzyme, and the
-phosphate undergoes a significant
change in its environment. The spectrum does not resemble that for
protonated GTP and therefore the
-phosphate clearly does not undergo
a simple protonation. The lack of detail in the low pH 180
difference spectrum is indicative of a heterogeneous environment for
the bound GTP. The protein unfolds at low pH; both its
- and
-structures are altered. The pH dependence of the amide I spectrum
of the Ras·GTP·Mg2+ complex reveals two unfolding
transitions: one that is reversible, with an apparent
pKa of 3.7, and a second, which is not reversible
and has a lower apparent pKa (2.6-1.7). The unfolding of the protein and the atypical difference spectrum indicate
that the Ras·GTP·Mg2+ complex, including its active
site, is heterogeneous at low pH.
The current study affirms several of the interpretations made in
previous work. The -,
-, and
-phosphate 31P-NMR
resonances of Ras·GTP·Mg2+ were shown to shift
concomitantly with a midpoint of 2.9, which was interpreted to be the
pKa of the
-phosphate (16, 19). It is not
possible, based solely on this correlation, to unambiguously assign the
physical basis of the chemical shift to protonation. For example, the
shift could be caused by a pH-dependent migration of the
Mg2+ ion on the tripolyphosphate chain or by a
protein structural change in the near environment of the
-phosphate.
In support of the previous interpretation, the current study provides
direct, site-specific confirmation of the facts that the
-phosphate
is not protonated in the Ras·GTP·Mg2+ complex and that
its pKa is shifted from 4.5 in solution to below 3.3 by the active site environment.
Consistent with our unfolding studies, the pH dependence of the
1H-NMR spectrum of Ras·GTP·Mg2+ shows that
the complex partially unfolds between pH 6.8 and 3.3 (16). In the pH
range 3.3-2.2, which includes the 2.9 midpoint, the 1H-NMR
changes are slight, suggesting that no major structural changes occur in the complex; however, our amide I data clearly show
that the secondary structure of the complex changes in this pH range. A
pH-dependent change in the structure of the
Ras·GTP·Mg2+ complex can explain the observed pH
dependence of the 31P chemical shift as well as the
simultaneous shift in the -,
-, and
-31P
resonances. Structural change could also be the basis for the pH rate
inactivation profiles used to construct the free energy plot
(
G
versus
pKa) described in the Introduction. The slope of the
free energy plot (i.e. 2.1) has been interpreted in several ways, including a protein isomerization that precedes hydrolysis (19).
This isomerization could, in fact, be the unfolding of the protein. The
pH-dependent unfolding of the Ras·GTP·Mg2+
complex provides a fully consistent, alternative interpretation of the
experimental support for the
-phosphate general base hypothesis and
thus raises the issue of whether this mechanism is operative in
GTPase-catalyzed reactions.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM35183 (to R. C.) and GM54469 (to T. S. L.) and National Science Foundation Grant MCB-9727439 (to R. C.).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,
Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx,
New York 10461-1926. Tel.: 718-430-2857; Fax: 718-430-8565; E-mail:
leyh@aecom.yu.edu.
Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009295200
1 H. Cheng, S. Sukal, T. S. Leyh, and R. Callender, accepted for publication.
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ABBREVIATIONS |
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The abbreviations used are: pD*, pH meter reading not corrected for D2O in the samples; FSD, Fourier spectral deconvolution.
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REFERENCES |
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