From the Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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The activity of GTP cyclohydrolase I is inhibited by (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and stimulated by phenylalanine through complex formation with GTP cyclohydrolase I feedback regulatory protein (GFRP). Gel filtration experiments as well as enzyme activity measurements showed that the number of subunits of GFRP in both the inhibitory and stimulatory complexes is equal to that of GTP cyclohydrolase I. Because GFRP is a pentamer and GTP cyclohydrolase I was shown here by cross-linking experiments to be a decamer, the results indicate that two molecules of a pentameric GFRP associate with one molecule of GTP cyclohydrolase I. Gel filtration analysis suggested that the complex has a radius of gyration similar to that of the enzyme itself. These observations support our model that one molecule of GFRP binds to each of the two outer faces of the torus-shaped GTP cyclohydrolase I. For formation of the inhibitory protein complex, both BH4 and GTP were required; the median effective concentrations of BH4 and GTP were 2 and 26 µM, respectively. BH4 was the most potent of biopterins with different oxidative states. Among GTP analogues, dGTP as well as guanosine 5'-O-(3'-thiotriphosphate) exhibited similar inducibility compared with GTP, whereas other nucleotide triphosphates had no effect. On the other hand, phenylalanine alone was enough for formation of the stimulatory protein complex, and positive cooperativity was found for the phenylalanine-induced protein complex formation. Phenylalanine was the most potent of the aromatic amino acids.
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INTRODUCTION |
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GFRP1 is a regulatory protein for GTP cyclohydrolase I (EC 3.5.4.16), the first and rate-limiting enzyme of the biosynthetic pathway of BH4 (1, 2). BH4 is synthesized from GTP by three enzymes (2). BH4 is an essential cofactor for tyrosine hydroxylase, a key enzyme for the biosynthesis of catecholamines; tryptophan hydroxylase, the first enzyme of the biosynthesis of serotonin; phenylalanine hydroxylase, the main enzyme of phenylalanine metabolism; and nitric-oxide synthase (2, 3). The biosynthesis of BH4 is thought to be tightly regulated with regard to the activities of those enzymes because the intracellular levels of BH4 are at or below the saturating levels for those enzymes (1). Despite the potential importance of the regulation, the molecular mechanisms are still unknown. A major advance in our understanding of this issue has been made by our identification of the regulatory protein GFRP (1). The discovery of GFRP and its effects on GTP cyclohydrolase I revealed how the biosynthesis of BH4 is linked to the activity of phenylalanine hydroxylase (1). In the presence of GFRP, BH4 inhibits the enzyme activity of GTP cyclohydrolase I, and the inhibition is reversed by phenylalanine. Elevated levels of phenylalanine thus stimulate the biosynthesis of BH4 to fill the cofactor requirement of phenylalanine hydroxylase, the activity of which is known to be also stimulated by phenylalanine (4). These mechanisms provide the explanation at a molecular level of several in vivo phenomena about the close relationship between the levels of BH4 and phenylalanine that were observed in normal individuals and patients with phenylketonuria (1, 5-7).
Rat GTP cyclohydrolase I was purified, and the kinetic analysis of the enzyme displayed positive cooperativity against GTP (8). This enzyme is a 300-kDa multimer composed of identical subunits with a subunit molecular mass of 25 kDa (8, 9), but its exact number of subunits has not yet been determined. Rat GFRP has been recently purified and cloned (10, 11). GFRP is a pentamer of identical subunits with a subunit molecular mass of 9.5 kDa (11).
The regulation of GTP cyclohydrolase I by GFRP occurs through ligand-induced complex formation between these two proteins, which is reversible (1). We showed by using an activity assay that GFRP is associated with GTP cyclohydrolase I both in the presence of BH4 and GTP and in the presence of phenylalanine, whereas no protein complex is formed in the absence of these ligand molecules. In particular, both GTP and BH4 are required for formation of the inhibitory protein complex, whereas phenylalanine alone is sufficient for formation of the stimulatory protein complex (1). Interestingly, phenylalanine reduces the positive cooperativity of GTP cyclohydrolase I in the presence of GFRP (1). However, detailed information regarding complex formation is lacking. We accordingly established a gel filtration method for analyzing complex formation. In this paper, we characterize the stoichiometry of both types of complexes and the ligand specificity for complex formation.
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EXPERIMENTAL PROCEDURES |
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Materials--
GTP was obtained from Yamasa (Chiba, Japan) and
U. S. Biochemical Corp., and GDP and GTPS were from Boehringer
Mannheim. BH4 was a generous gift from the Suntory
Institute for Medicinal Research and Development (Gunma, Japan).
BH2 was purchased from Schircks Laboratories (Jona,
Switzerland). L-Biopterin and dGTP were from Sigma.
Recombinant rat GTP cyclohydrolase I and GFRP were prepared as
described previously (1, 11).
Determination of Extinction Coefficients of GTP Cyclohydrolase I
and GFRP--
Absorbance spectra of GTP cyclohydrolase I and GFRP were
measured with a Perkin-Elmer Lambda 2 spectrophotometer. The extinction coefficients of GTP cyclohydrolase I and GFRP were determined to be
Em,280 = 0.72 and 1.23 (mg/ml)-1 cm-1, respectively, based on the
mass data obtained by amino acid analysis. Taking the subunit molecular
weights of GTP cyclohydrolase I and GFRP as 25,784 and 9946 from the
corresponding DNA sequences (1, 9, 11), we calculated the molar
extinction coefficients to be M,280 = 1.86 × 104 and 1.22 × 104
M-1 cm-1, respectively. All the
molar concentrations of GFRP and GTP cyclohydrolase I used in this
paper are expressed as those of their subunits.
Cross-linking of GTP Cyclohydrolase I-- Cross-linking reactions were performed in 500 µl of 200 mM triethanolamine HCl buffer (pH 8.5) containing 0.1 mM dithiothreitol at a GTP cyclohydrolase I concentration of 0.1 mg/ml. The reaction mixture was prepared on ice. To the mixture was added freshly prepared dimethyl suberimidate to concentrations varying from 0.05 to 1.0 mg/ml. The mixture was incubated at room temperature for 3 h. Protein was precipitated using a deoxycholate/Cl3CCOOH procedure (12). The precipitates were washed with ethyl ether to remove Cl3CCOOH and dissolved in electrophoresis sample buffer. Cross-linked protein was resolved by electrophoresis on 5% (w/v) polyacrylamide gels in the presence of SDS (13). After electrophoresis, protein was stained with Coomassie Brilliant Blue R-250 (Bio-Rad).
Assay of GTP Cyclohydrolase I Activity-- The activity of GTP cyclohydrolase I was assayed as described (8, 14). The reaction mixture (50 µl) contained 50 mM Hepes-KOH buffer (pH 7.2), 0.2 M KCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 200 µM GTP, and recombinant GTP cyclohydrolase I. The reaction was carried out at 25 °C for 30 min.
Gel Filtration-- Assay for the complex formation between GFRP and GTP cyclohydrolase I was performed using gel filtration, which enabled us to separate the complex formed from free GFRP. Protein samples were separated on a 1 × 30-cm column of Superdex 75 (Amersham Pharmacia Biotech) or a 7.8 mm × 30-cm column of TSK G3000SWXL (Tosoh, Tokyo, Japan). The columns were equilibrated with 50 mM Hepes-KOH buffer (pH 7.2) containing 0.2 M KCl, 1 mM EDTA, 1 mM dithiothreitol, and ligands indicated in the figure legends. Elution was performed at a flow rate of 0.8 ml/min for Superdex 75 and 1.0 ml/min for G3000SWXL at room temperature. The eluate was monitored at 280 nm. The peak heights of each GFRP and GTP cyclohydrolase I were linear at least in the range of protein injected between 0.1 and 0.8 nmol when the proteins were separately filtered through the gel resins in the presence of phenylalanine or a combination of BH4 and GTP as well as in the absence of any ligands.
Quantitation of Proteins Separated by Gel Electrophoresis Using Coomassie Brilliant Blue-- Fractions obtained from gel filtration were collected, and proteins were precipitated as described above and dissolved in electrophoresis sample buffer. Protein was resolved by electrophoresis on 16.5% (w/v) polyacrylamide gels in the presence of SDS as described by Laemmli (15). Quantitation of proteins separated by electrophoresis was done as described (16). Briefly, after electrophoresis, protein was fixed and stained with Coomassie Brilliant Blue G-250 (SERVA Blue G, Boehringer Ingelheim Bioproducts, Heidelberg, Germany) as described. After destaining of the gel, the protein bands were cut out of the gel. Dye was eluted from the gel pieces with 3% SDS, and absorbance at 610 nm was measured. Data were first corrected for background staining. Then, the corrected values of A610 for GFRP were deduced by the following equation: A610 measured for GFRP × (A610 measured for GTP cyclohydrolase I in Fig. 5 (lane 1) (no GFRP contained)/A610 measured for GTP cyclohydrolase I in the same lane of the GFRP measured).
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RESULTS AND DISCUSSION |
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Subunit Number of Rat GTP Cyclohydrolase I
Escherichia coli GTP cyclohydrolase I was shown to be a decamer by crystallographic studies (17). Considering the high sequence similarity between E. coli (18) and rat (9) GTP cyclohydrolase I and their similar native molecular weights (8, 17), we hypothesized that rat GTP cyclohydrolase I also was a decamer. To determine the subunit number of GTP cyclohydrolase I, we performed cross-linking experiments using dimethyl suberimidate, a homobifunctional imidoester that is able to cross-link lysine residues. The cross-linking reagent was used at a variety of concentrations to obtain proteins cross-linked to variable extents. The cross-linked proteins were then analyzed by SDS-polyacrylamide gel electrophoresis under the conditions established by Weber and Osborn (13). As shown in Fig. 1, significant intersubunit cross-linking of GTP cyclohydrolase I was obtained at a dimethyl suberimidate concentration of 0.05 mg/ml. As higher concentrations of dimethyl suberimidate were used, the protein was further cross-linked. At a dimethyl suberimidate concentration of 1.0 mg/ml, the proteins were almost completely cross-linked and converged to one species with a molecular mass of 280 kDa. When the estimated molecular masses of the protein species found in the gel were plotted against subunit number, a linear relationship was obtained, and the number of the largest species was 10. This finding demonstrates that GTP cyclohydrolase I is a decamer.
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Inhibitory Complex Formation
Dose Dependence of GFRP for Its BH4-dependent Inhibitory Action on GTP Cyclohydrolase I Activity-- To determine the amount of GFRP required to totally inhibit GTP cyclohydrolase I, we examined the effect of varying amounts of GFRP on the activity of a fixed amount of GTP cyclohydrolase I (0.23 µM) in the presence of a saturating concentration of BH4 (Fig. 2). Because the Kd value for the association between GFRP and GTP cyclohydrolase I is in the nanomolar range (1), the concentrations used were saturating for formation of the protein complex. As shown in Fig. 2, the activity of GTP cyclohydrolase I was inhibited in a GFRP dose-dependent manner and became totally inhibited when the GFRP concentration was above 0.25 µM. This result indicates that the same number of GFRP as GTP cyclohydrolase I in terms of subunits is necessary and sufficient for total inhibition of the enzyme activity. Because GFRP is a pentamer (11) and GTP cyclohydrolase I is a decamer as shown above, this finding indicates that two molecules of GFRP are required for the total inhibition of one molecule of GTP cyclohydrolase I.
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Protein Stoichiometry of the Inhibitory Complex Formation between GTP Cyclohydrolase I and GFRP-- The kinetic experiments suggested that two molecules of GFRP were associated with one molecule of GTP cyclohydrolase I. To physically determine the number of GFRP associated with GTP cyclohydrolase I, we used a gel filtration method by which the complex was separated from free GFRP.
It should be noted that, when each protein was separately filtered through the resin in the presence of either or both BH4 and GTP (data not shown), both proteins eluted from the gel resin at the same volume as those in the absence of ligands (Fig. 3, A1 and A2), indicating that these ligands induced neither dissociation into subunits of smaller number nor further association of each multimer. Thus, GFRP and GTP cyclohydrolase I are stable in terms of subunit assembly under the gel filtration conditions used. These properties of the proteins made the gel filtration experiments feasible for assessing complex formation.
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Behavior of the Complex on Gel Filtration--
As described above,
the elution volume of the complex from a TSK G3000SWXL
column was almost the same as that of GTP cyclohydrolase I. Since the
elution position of the complex from the column was close to the void
volume, it was outside the given linear estimation of molecular masses.
Thus, we performed gel filtration using Superose 6 resins, which gave a
straight line over the molecular mass range of 150-669 kDa. GTP
cyclohydrolase I and the complex were eluted from this column at close
volumes, which corresponded to the relative molecular masses of 300 and
313 kDa, respectively, when compared with those of bovine thyroglobulin
(669 kDa), sweet potato -amylase (200 kDa), and yeast alcohol
dehydrogenase (150 kDa). This observation suggests that the radius of
gyration of GTP cyclohydrolase I did not change even after two
molecules of GFRP with a molecular mass of 50 kDa were bound to the
enzyme. Considering that the shape of the corresponding E. coli GTP cyclohydrolase I is a torus formed by face-to-face
association of two pentamers with dimensions of 65 × 100 Å (17),
we infer that the structure of the complex is such that one molecule of
the GFRP pentamer binds to each of the two outer faces of the pentamer
of GTP cyclohydrolase I; in other words, GTP cyclohydrolase I is
sandwiched between GFRPs. Thus, the complex might behave on gel
filtration as though the apparent diameter was similar to that of
GTP cyclohydrolase I.
Dose Dependence and Specificity of BH4 and GTP for Their Inducibility of Complex Formation-- By using the gel filtration method, we examined the effect of varying concentrations of BH4 on complex formation between GFRP and GTP cyclohydrolase I in the presence of GTP. As shown in Fig. 6A, the protein complex was formed in a BH4-dose dependent manner. The median effective concentration (EC50) of BH4 was estimated from the graph to be 2 µM. This value is equal to the EC50 value of BH4 (2 µM) that was kinetically determined for the inhibition of GTP cyclohydrolase I activity at 100 µM GTP (11). This finding suggests that the physical association of GFRP with GTP cyclohydrolase I is tightly coupled to the inhibition of enzyme activity.
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Stimulatory Complex Formation
Dose Dependence of GFRP for Its Phenylalanine-dependent Stimulatory Action on GTP Cyclohydrolase I Activity-- In the presence of GFRP, phenylalanine changes the kinetic nature of GTP cyclohydrolase I from sigmoidal to hyperbolic in a dose-dependent manner; in other words, phenylalanine stimulates the enzyme activity when GTP concentrations are below saturation (1, 11). To determine the amount of GFRP required for producing the maximum effect of phenylalanine, we examined the effect of varying concentrations of GFRP on the activity of GTP cyclohydrolase I at a non-saturating GTP concentration of 20 µM in the presence of 1 mM phenylalanine. With this phenylalanine concentration, the substrate velocity curve for GTP cyclohydrolase I fully changes from sigmoidal to hyperbolic (1, 11). The concentration of GTP used (20 µM) is a concentration at which the activity of GTP cyclohydrolase I increases ~4-fold in the presence of a saturating amount of GFRP and 1 mM phenylalanine (11). Under these conditions, GFRP stimulated the activity of GTP cyclohydrolase I in a dose-dependent manner and leveled off at a concentration of 0.2 µM, which is the same concentration as that of GTP cyclohydrolase I contained in the reaction mixture (Fig. 7). This result indicates that, as in the case of the GFRP-dependent inhibition of GTP cyclohydrolase I, an equimolar number of GFRP subunits is necessary for the total activation of GTP cyclohydrolase I activity, indicating that two molecules of GFRP are required for the total activation of one molecule of GTP cyclohydrolase I.
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Protein Stoichiometry of the Stimulatory Complex Formation between GTP Cyclohydrolase I and GFRP-- To physically determine the protein stoichiometry of the stimulatory complex formed between GFRP and GTP cyclohydrolase I, we performed similar gel filtration experiments as for the inhibitory complex. When each protein was separately filtered through the resin in the presence of phenylalanine, both GFRP and GTP cyclohydrolase I eluted from the gel resin at the same volumes as they did in the absence of any ligands, indicating that phenylalanine did not alter the association state of the subunits of either protein. This fact was a prerequisite for the following experiments.
With regard to the phenylalanine-dependent complex formation, we obtained chromatograms (Figs. 3B and 4B1) that were similar to those obtained for the inhibitory complex (Figs. 3A and 4A1). It should be noted that the increase in the peak height of GTP cyclohydrolase I was not a linear function of the amount of GFRP (Fig. 4B1). However, the quantitative analysis of proteins in the peak fraction by gel electrophoresis showed that the amount of GFRP associated with GTP cyclohydrolase I increased linearly and leveled off at the same molar amount as that of GTP cyclohydrolase I (Fig. 4B2). These findings indicate that phenylalanine also induces association of two molecules of pentameric GFRP with one molecule of decameric GTP cyclohydrolase I.Dose Dependence and Specificity of Phenylalanine for Its
Inducibility of Complex Formation--
We examined the effect of
varying concentrations of phenylalanine on complex formation. As shown
in Fig. 8, the resulting curve was
sigmoidal, indicating a positive cooperativity of phenylalanine in its
ability to induce the association between GFRP and GTP cyclohydrolase
I. By assuming that the binding of phenylalanine to the proteins
induces formation of the protein complex, we treated the saturation
data as a binding curve and converted it into a Hill plot:
log[A/(1 -
A)] versus
log[phenylalanine], where
A was the measured change in
the amount of GFRP bound to GTP cyclohydrolase I. The resulting plot
was linear in its middle portion with a slope nH
(Hill coefficient) of 2.0. The K0.5 of
phenylalanine was estimated from the graph to be 65 µM.
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Conclusion-- We presented two lines of evidence, kinetic as well as physicochemical, demonstrating that two molecules of GFRP physically bind to one molecule of GTP cyclohydrolase I. The protein stoichiometry turned out to be the same for both types of protein complex induced by phenylalanine or by a combination of BH4 and GTP. Because GFRP is a pentamer (11) and GTP cyclohydrolase I is a decamer as shown by the cross-linking experiments presented in this paper, the complex thus contains equal numbers of subunits of GFRP and GTP cyclohydrolase I. This fact is consistent with the model we previously proposed regarding the structure of the protein complex, partly by assuming the general symmetric structure of protein complexes (11). In that model, we proposed that one molecule of GFRP binds to each face of a disc-like structure of GTP cyclohydrolase I, which is formed by face-to-face association of two pentamers. Such a structure of GTP cyclohydrolase I was revealed by x-ray crystallographic analysis of E. coli GTP cyclohydrolase I (17). Because the E. coli and rat enzymes exhibit considerable sequence similarity and have the same number of subunits, it is likely that rat GTP cyclohydrolase I has a three-dimensional arrangement of subunits similar to that of the bacterial enzyme.
The specificity of the biopterins and aromatic amino acids with regard to induction of protein complex formation was similar to that for the catalytic effects, i.e. BH4 and phenylalanine were the most effective. Interestingly, the ability of nucleotides to induce protein complex formation was highly specific in terms of the structure of the base, but was nonspecific in terms of the hydroxylation state of the ribose moiety at the 2'-position and of the structure of the third position of phosphate. Positive cooperativity was found with respect to phenylalanine in the association of GFRP with GTP cyclohydrolase I and also in the GFRP-mediated stimulation of GTP cyclohydrolase I activity, whereas no cooperative phenomenon was observed for either BH4 and GTP in the inhibitory complex formation. The positive cooperativity means that GTP cyclohydrolase I activity increases sharply with only a relatively small increase in phenylalanine concentration. This function may operate physiologically as BH4 is sufficiently supplied to phenylalanine hydroxylase when the plasma phenylalanine concentration increases. Finally, when all the physical data presented herein on protein complex formation were compared with the corresponding kinetic data, a close relationship was found between the physical association of GFRP with GTP cyclohydrolase I and the change in the activity of GTP cyclohydrolase I. It is thus established that the regulation of GTP cyclohydrolase I by GFRP occurs through a mechanism of protein complex formation. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Sidney M. Morris for critical reading of the manuscript, Dr. John D. Hempel for amino acid analysis, and Drs. Timothy R. Billiar and Richard L. Simmons for continuous support.
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FOOTNOTES |
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* This work was supported in part by the University of Pittsburgh Competitive Medical Research Fund (to K. H.) and by National Institutes of Health Research Grant DK51257 (to K. H.).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 Surgery,
University of Pittsburgh, Kaufman Bldg., Suite 300, 3471 Fifth Ave.,
Pittsburgh, PA 15213. Tel.: 412-648-6774; Fax: 412-692-2520.
The abbreviations used are:
GFRP, GTP
cyclohydrolase I feedback regulatory protein; BH4, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterinBH2, 7,8-dihydrobiopterinGTPS, guanosine
5'-O-(3'-thiotriphosphate).
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REFERENCES |
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