gamma -Phosphate Protonation and pH-dependent Unfolding of the Ras·GTP·Mg2+ Complex

A VIBRATIONAL SPECTROSCOPY STUDY*

Hu Cheng, Sean Sukal, Robert Callender, and Thomas S. LeyhDagger

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 gamma -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 gamma -phosphate is not protonated at pH >=  3.3, indicating that the active site decreases the gamma -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-gamma -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 gamma -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

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 gamma -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 gamma -phosphate general base theory, which hypothesizes that the gamma -phosphate of GTP, rather than an amino acid R-group, is the general base.

The idea that the gamma -phosphate might be the general base in GTPase-catalyzed hydrolysis reactions was described first in a computational study that calculated that the hydrolysis Delta GDagger 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 alpha -, beta -, and gamma -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 (Delta GDagger 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 gamma -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 gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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 >= 10% of the soluble cellular fraction (SDS-polyacrylamide gel electrophoresis).

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 -70 °C.

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/[gamma -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.

The Synthesis of [gamma -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 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 [gamma -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.

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 -70 °C. TLC on polyethyleneimine F-cellulose plates showed that the [gamma -18O3]GTP was more than than 95% pure.

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 [gamma -18O3]GTP.

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 [gamma -18O3]GTP was added. Nucleotide exchange was complete after 30 min at 25 ± 2 °C.

Sample Preparation for gamma -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.

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-1 and 2000 cm-1 is nulled. The contribution from vapor was also removed by subtraction.

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-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

The validity of the gamma -phosphate general base hypothesis rests on the proposition that the gamma -phosphate of Ras-bound GTP partially abstracts a proton from the nucleophilic water, making it more hydroxide-like, thereby activating it to attack the gamma -phosphate and cleave the beta ,gamma -bond of GTP. Experimental support for this hypothesis is provided by 31P-NMR pH titration studies of Ras·GTP·Mg2+ in which the alpha -, beta -, and gamma -31P resonance positions shifted concomitantly at a pH midpoint of 2.9. This midpoint was interpreted as the pKa of the GTP gamma -phosphate in the Ras·GTP complex (16). In solution, the alpha -, beta -, and gamma -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.

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-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 gamma -phosphate undergoes as the pH passes through the 2.9-midpoint, the pH dependence of the vibrational spectrum of the gamma -phosphate of GTP in the Ras·GTP·Mg2+ complex was studied using IR spectroscopy.

The vibrational spectrum of the Ras·GTP·Mg2+ complex is quite complex, and identifying the stretch band of gamma -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 gamma -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

To aid in assigning the Ras·GTP·Mg2+ difference spectra and to provide comparisons between the active site and solution behavior of the gamma -phosphate, the difference spectra for the gamma -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.


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Fig. 1.   pH dependence of the GTP gamma -Phosphate IR Difference Spectra. A, the GTP·Mg2+ solution spectra. The sample composition was as follows: [gamma -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·[gamma -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) gamma -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 gamma -phosphate and the active site (27, 28).1 These similarities strongly support the notion that the gamma -phosphate is dianionic at the active site. The spectrum begins to change at pH 3.3, indicating that the gamma -phosphate remains largely unprotonated at this pH. Thus, the active site of Ras has decreased the pKa of the gamma -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 gamma -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 gamma -phosphate. Whether or not gamma -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 gamma -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 cm-1) 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 alpha -helix and absorbance at 1635 and 1675 cm-1 to the low (intense) and high (weak) frequency components of the beta -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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Fig. 2.   The pH dependence of the amide I spectra of Ras·GTP·Mg2+ and Ras·GDP·Mg2+. A and B, the pH dependence of the amide I spectra of Ras·GTP·Mg2+ and Ras·GDP·Mg2+, respectively. C and D, Fourier spectral deconvolutions of the data shown in A and B, respectively. Samples were prepared, acquired, and deconvoluted as described under "Materials and Methods."

Ras contains large sections of beta -sheet and alpha -helix, and, as expected, the amide I region of the FSD spectrum of the Ras-nucleotide complexes near neutrality show appropriate amplitude in the alpha -helical (1656 cm-1) and beta -sheet (1635 and 1674 cm-1) regions. Low pH denatures both the alpha -helical and beta -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 gamma -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).


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Fig. 3.   The pH-dependent unfolding of Ras·GTP·Mg2+ and Ras·GDP·Mg2+. The pH titrations of the amide spectral changes that occur in the Ras·GTP·Mg2+ () and Ras·GDP·Mg2 (black-triangle) complexes are shown. The apparent pKa values were obtained by fitting the data using the Henderson-Hasselbalch equation for one (black-triangle) or two () protons. The best fits are represented by the solid lines. The construction of the plots is discussed under "Materials and Methods."

Conclusions-- The difference spectrum for the deprotonated GTP gamma -phosphate in solution resembles that for the Ras·GTP·Mg2+ complex at pH >=  3.3 and differs markedly from the protonated gamma -phosphate spectrum. It is clear from this and other vibrational studies (26-28) that the GTP gamma -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 gamma -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 gamma -phosphate undergoes a significant change in its environment. The spectrum does not resemble that for protonated GTP and therefore the gamma -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 alpha - and beta -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 alpha -, beta -, and gamma -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 gamma -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 gamma -phosphate. In support of the previous interpretation, the current study provides direct, site-specific confirmation of the facts that the gamma -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 alpha -, beta -, and gamma -31P resonances. Structural change could also be the basis for the pH rate inactivation profiles used to construct the free energy plot (Delta GDagger 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 gamma -phosphate general base hypothesis and thus raises the issue of whether this mechanism is operative in GTPase-catalyzed reactions.

    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.

Dagger 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.

    ABBREVIATIONS

The abbreviations used are: pD*, pH meter reading not corrected for D2O in the samples; FSD, Fourier spectral deconvolution.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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