Deamidations in Recombinant Human Phenylalanine Hydroxylase

IDENTIFICATION OF LABILE ASPARAGINE RESIDUES AND FUNCTIONAL CHARACTERIZATION OF ASN right-arrow ASP MUTANT FORMS*

Raquel Negrão CarvalhoDagger , Therese SolstadDagger , Elisa Bjørgo§, João Filipe BarrosoDagger , and Torgeir FlatmarkDagger

From the Dagger  Department of Biochemistry and Molecular Biology and the Proteomic Unit, University of Bergen, N-5009 Bergen and the § Biotechnology Center of Oslo, University of Oslo, 0317 Blindern, Oslo, Norway

Received for publication, December 2, 2002, and in revised form, January 21, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recombinant human phenylalanine hydroxylase (hPAH) expressed in Escherichia coli for 24 h at 28 °C has been found by two-dimensional electrophoresis to exist as a mixture of four to five molecular forms as a result of nonenzymatic deamidation of labile Asn residues. The multiple deamidations alter the functional properties of the enzyme including its affinity for L-phenylalanine and tetrahydrobiopterin, catalytic efficiency, and substrate inhibition and also result in enzyme forms more susceptible to limited tryptic proteolysis. Asn32 in the regulatory domain deamidates very rapidly because of its nearest neighbor amino acid Gly33 (Solstad, T., Carvalho, R. N., Andersen, O. A., Waidelich, D., and Flatmark, T. (2003) Eur. J. Biochem., in press). Matrix-assisted laser desorption/ionization time of flight-mass spectrometry of the tryptic peptides in the catalytic domain of a 24-h (28 °C) expressed enzyme has shown Asn376 and Asn133 to be labile residues. Site-directed mutagenesis of nine Asn residues revealed that the deamidations of Asn32 and Asn376 are the main determinants for the functional and regulatory differences observed between the 2- and 24-h-induced wild-type (wt) enzyme. The Asn32 right-arrow Asp, Asn376 right-arrow Asp, and the double mutant forms expressed for 2 h at 28 °C revealed qualitatively similar regulatory properties as the highly deamidated 24-h expressed wt-hPAH. Moreover, deamidation of Asn32 in the wt-hPAH (24 h expression at 28 °C) and the Asn32 right-arrow Asp mutation both increase the initial rate of phosphorylation of Ser16 by cAMP-dependent protein kinase (p < 0.005). By contrast, the substitution of Gly33 with Ala or Val, both preventing the deamidation of Asn32, resulted in enzyme forms that were phosphorylated at a similar rate as nondeamidated wt-hPAH, even on 24-h expression. The other Asn right-arrow Asp substitutions (in the catalytic domain) revealed that Asn207 and Asn223 have an important stabilizing structural function. Finally, two recently reported phenylketonuria mutations at Asn residues in the catalytic domain were studied, i.e. Asn167 right-arrow Ile and Asn207 right-arrow Asp, and their phenotypes were characterized.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phenylalanine hydroxylase (PAH,1 phenylalanine 4-monooxygenase, EC 1.14.16.1) is a non-heme iron monooxygenase that catalyzes the hydroxylation of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr) in the presence of the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4biopterin) and dioxygen. Mutations in the human enzyme (hPAH) leading to altered kinetic properties or reduced stability of the enzyme are associated with the autosomal recessive disorder phenylketonuria (PKU). hPAH isolated from liver (2) and as recombinant enzyme expressed in Escherichia coli (3, 4) has been found to exist as a mixture of 4-5 molecular forms with the same apparent subunit molecular mass, but with different isoelectric points (pI). Isoelectric focusing and two-dimensional electrophoresis of recombinant hPAH expressed in E. coli for 24 h at 28 °C revealed five components of decreasing staining intensity and decreasing pI (denoted hPAH I-V). This microheterogeneity was shown to be the result of nonenzymatic deamidations of Asn residues (1, 4). Mainly one band with the highest pI (hPAH I) was, however, detected after a short induction period of 2 h at 28 °C, and this form was considered to represent the newly synthesized and most native, nondeamidated form of the enzyme. Thus, the microheterogeneity pattern is highly dependent on the induction time with IPTG in E. coli (4). Due to the relatively high rate of deamidation, the labile amide-containing residues have been considered to be Asn residues (4), and this conclusion has recently been confirmed by the demonstration of iso-Asp in several tryptic peptides of the highly deamidated full-length wt-hPAH (1). In the deamidation reaction Asn is converted to Asp and iso-Asp in a variable ratio, sometimes with iso-Asp as the main product (5, 6). The rate of Asn deamidation in proteins and peptides has been shown to be dependent on pH, temperature, and ionic strength (7) and on intrinsic factors like the nearest neighbor amino acids, particularly the residue in the (n + 1) position (8-10). The deamidation proceeds via a cyclic succinimide intermediate (6), and with glycine in the n + 1 position the rate of deamidation is unusually rapid, whereas all the other 19 amino acid residues are more sterically hindered in the formation of the cyclic intermediate (11). Moreover, the rate of deamidation is also determined by the protein secondary and tertiary structure because the necessary flexibility for the formation of the cyclic intermediate may be limited in proteins (12). The fact that deamidation is an ordered process adds to the complexity, as structural changes induced by deamidation of one residue may influence further sequential deamidation reactions (reviewed in Ref. 13). The rate of deamidation in model peptides has also been shown to be inversely proportional to the extent of alpha -helicity which suggests that Asn deamidation occurs preferentially in nonhelical structural elements (14). The deamidation of Asn introduces an additional negative charge (Asp or iso-Asp) that may result in significant conformational changes of the protein as well as altered functional properties. A difference in functional properties was first described for the multiple deamidated forms of bovine heart cytochrome c, i.e. a deactivation (15), and more recently also shown to be the case for wt-hPAH, but in this case with an activation of the enzyme (4). A comparison of the catalytic properties of nondeamidated and highly deamidated enzyme preparations revealed that the catalytic efficiency (kcat/[S]0.5) and the apparent affinity for the substrate (L-Phe) were higher for the multiple deamidated forms than for the nondeamidated enzyme (4). Moreover, in enzyme kinetic studies the deamidated forms of hPAH revealed a higher Hill coefficient than the nondeamidated enzyme (h = 1.9 versus 1.5) for the cooperative binding of L-Phe as well as a more pronounced substrate inhibition.

The sequence of hPAH contains 16 Asn residues, but only about 4-5 are expected to be involved in the deamidations that occur in the enzyme on 24-h expression in E. coli (4). On the basis of a computer algorithm that estimates the deamidation rates by taking into account the nearest neighbor amino acids and the three-dimensional structure of the protein (16), the relative deamidation rates of all the Asn residues in hPAH have been predicted (17). We have recently verified that Asn32 (followed by a glycine residue), Asn28, and Asn30 in a loop region of the N-terminal autoregulatory sequence (residues 19-33) of wt-hPAH are among the susceptible residues (1). On the basis of the computational method two other labile residues have been predicted to be located in the catalytic domain structure, i.e. Asn133 and Asn376 (17). Here we present experimental data verifying this prediction based on MALDI-TOF mass spectrometry of tryptic peptides and site-directed mutagenesis. Mutagenesis was also used to characterize the structural and functional effects of single Asn right-arrow Asp substitutions at 8 alternative positions in the catalytic domain. Moreover, the functional effects of deamidation of Asn32 are further studied in mutant forms with special reference to its effect on the rate of phosphorylation of Ser16 by PKA, used as a conformational probe.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis-- The mutations in the catalytic domain were introduced into the wt-hPAH cDNA by PCR-based site-directed mutagenesis (18) using the pMAL-hPAH vector, containing the factor Xa cleavage site (New England Biolabs) as a template (19), and the specific oligonucleotide primers listed in Table I. The target sequences for mutagenesis were the restriction endonuclease fragments XbaI/BamHI (N133D, N167D, and N167I), XhoI/BamHI (N207D), XhoI/AflII (N223D), and BamHI/AflII (N376D, N393D, N401D, and N426D). The three mutations in the regulatory domain and the double mutant N32D/N376D were introduced into the pMAL-hPAH expression system containing the enterokinase cleavage site (D4K) (New England Biolabs) using the QuikChangeTM site-directed mutagenesis kit (Stratagene) and the specific oligonucleotides listed in Table I. Authenticity of mutagenesis was verified by DNA sequencing using the malE and 13B oligonucleotides (20) and the Big DyeTM Terminator Ready Reaction Mix (PerkinElmer Life Sciences) in an ABI PrismTM 377 DNA Sequencer (PerkinElmer Life Sciences). MWG-Biotech AG provided primers for mutagenesis and sequencing. Some of the mutants were also verified by MALDI-TOF mass spectrometry of the isolated recombinant enzyme (see "Results").


                              
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Table I
Oligonucleotides used for PCR-based mutagenesis in the catalytic and regulatory domains
Mismatched nucleotides are shown in boldface.

Expression and Purification of the Enzymes-- The wild-type and the mutant forms of hPAH were expressed as fusion proteins in E. coli (TB1 cells) using the maltose-binding protein as a fusion partner (21). Cells were grown at 37 °C, and expression was induced at 28 °C by the addition of 1 mM isopropyl thio-beta -D-galactoside (IPTG). The cells expressing wt-hPAH were harvested after 2 and 24 h of induction, and the mutant forms were isolated after an induction period of 2 h, except where otherwise stated. After protein purification by affinity chromatography (amylose resin, 4 °C), the fusion proteins were cleaved for 5 h at 4 °C by the restriction protease enterokinase (Invitrogen) using 4 units of protease/mg of fusion protein or by factor Xa (from Protein Technology ApS, Denmark) using a protease to substrate ratio of 1:200 (by mass). The tetrameric forms were isolated by size-exclusion chromatography at 4 °C using a HiLoad Superdex 200 HR column (1.6 × 60 cm), prepacked from Amersham Biosciences.

Mass Spectrometry-- Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) spectra of tryptic peptides were acquired on a 4700 Proteomic Analyzer (Applied Biosystems Inc.) in the reflectron positive-ion mode and were externally mass calibrated. The instrument was supplied with a software tool that uses a scanning algorithm to isotopically deconvolute the mass spectra (Sierra Analytics, Modesto, CA). The deconvolution method was particularly useful to detect labile Asn residues in a peptide because deamidation of Asn to Asp/iso-Asp increases the monoisotopic mass ([M + H]+) of the peptide by only 1 Da.

Assay of hPAH Activity-- The hPAH activity was assayed as described (21), but the catalase concentration was 0.1 µg/µl, and the enzyme was activated by prior incubation (5 min) with L-Phe. Moreover, 0.5% (w/v) bovine serum albumin was included in the reaction mixture to stabilize the diluted purified enzyme. The enzyme source was either isolated cleaved tetrameric form or purified tetrameric fusion protein in the case of the N167I mutant, and the reaction time was 1 min. The steady-state kinetic data were analyzed by nonlinear regression analysis using the SigmaPlot® Technical Graphing Software and the modified Hill equation of LiCata and Allewell (22) for cooperative substrate binding as well as substrate inhibition (4, 23). In some experiments 1 mM L-Phe was added either at the start of the preincubation period or together with 75 µM pterin cofactor (H4biopterin) at the initiation of the hydroxylation reaction. A 3-min time course was then followed in order to study the effect of preincubation with L-Phe on the specific activity of the wild-type and mutant forms of hPAH.

Proteolysis by Trypsin-- Limited proteolysis of the wild-type and mutant tetrameric forms was performed at 25 °C and a protease to hPAH ratio of 1:200 (by mass). At timed intervals (0-60 min) aliquots were removed and subjected to SDS-PAGE (24), and the band corresponding to the full-length subunit was quantified. For peptide analysis by mass spectrometry and reversed-phase chromatography, tryptic proteolysis of hPAH was performed in 20 mM Na-HEPES buffer, pH 7.0, at 30 °C for 2 h at a trypsin to substrate ratio of 1:10 (by mass). Soybean trypsin inhibitor was added at the end with a protease to inhibitor ratio of 1:1.5 (by mass).

Reversed-phase Chromatography-- The mutant tetrameric enzymes were 32P-labeled by phosphorylation with PKA (see below), proteolyzed by trypsin, and incubated at 37 °C up to 32 h to determine the rate of deamidation of labile Asn residues in the phosphopeptide(s) (1). At different time points the incubated peptide mixture was subjected to reversed-phase chromatography using a ConstaMetric gradient System (Laboratory Data Control) and a 4.6-mm × 10-cm Hypersil ODS C18 column (Hewlett-Packard) fitted with a 2-cm guard column of the same material. Solvent A was 50 mM ammonium acetate (pH 8.0) and solvent B 50 mM ammonium acetate in 70% (v/v) acetonitrile (pH 8.0). A linear gradient of 10-50% solvent B was used at a flow rate of 1 ml/min for 60 min (1). Samples were collected every 15 s, and the elution pattern of phosphopeptides was analyzed by liquid scintillation counting and resolved into individual components, using the Peak Fit software program (SPSS Inc., Chicago); the "auto-fit II-Residuals" was used with the confidence level set at >= 95%.

Phosphorylation by PKA-- The time course for the phosphorylation by PKA of Ser16 in tetrameric wt-hPAH and its mutant forms (5 µM subunit) was performed at 30 °C in a medium containing 15 mM Na-HEPES, 3 mM dithiothreitol, 0.03 mM EDTA, 0.1 mM EGTA, 10 mM magnesium acetate, 60 µM ATP ([gamma -32P]ATP from Amersham Biosciences), and 50 nM of PKA catalytic subunit. The initial rate of phosphorylation and its mean ± S.E. was estimated by nonlinear regression analysis of the 15 individual data points collected within the first 2.5 min (three data points at each time point) (25). The Student's t test was used for the statistical analysis.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression and Purification of wt-hPAH and Its Mutant Forms-- The introduction of extra negative charges in wt-hPAH as a result of nonenzymatic deamidation during a 24-h expression period at 28 °C in the E. coli pMal system has been shown to increase the solubility and recovery of recombinant wt-hPAH relative to the 2-h expressed enzyme (4). The expression of the Asn right-arrow Asp mutant forms of hPAH resulted in a variable but generally increased yield of fusion proteins (relative to wt-hPAH) after an IPTG induction period of only 2 h at 28 °C. Size-exclusion chromatography of the affinity-purified mutant fusion proteins (data not shown) revealed a pattern of oligomerization (tetramers dimers) similar to that of wt-hPAH (21, 26, 27). Moreover, after their cleavage by restriction protease, no significant amount of aggregated enzyme forms was observed in the size-exclusion chromatographic profiles. One-dimensional SDS-PAGE analysis of all the mutant forms revealed the same relative electrophoretic mobility as for the wt-hPAH protomer, both as uncleaved and cleaved fusion protein (data not shown).

Mass Spectrometry-- To identify the labile Asn residues in wt-hPAH and Delta N-(1-102)/Delta C-(429-452)-hPAH forms, their tryptic peptides were analyzed by MALDI-TOF mass spectrometry. For wt-hPAH expressed for 2 h at 28 °C, the matching peptides covered 11 of the total 16 Asn residues in the protein, and on isotopic deconvolution of the mass spectra (28) all the peptides revealed a single monoisotopic mass peak ([M + H+]) (Table II). The peptides containing Asn8, Asn58, Asn61, Asn223, and Asn401 were, however, not recovered in the mass spectrum but were not among the predicted labile residues (17). On expression of wt-hPAH for 24 h at 28 °C, the 28-residue peptide (residues 15-42), containing Asn28, Asn30, and the most labile Asn32, revealed three additional monoisotopic peaks at the m/z 3107.47, 3108.47, and 3109.48 in addition to the 3106.51 peak of Table II (1). Moreover, the mass spectrum of the 25-residue peptide (Thr372-Lys396) containing Asn376 revealed on deconvolution an additional monoisotopic peak at m/z 2913.36, i.e. 1 Da higher than the mass peak at m/z 2912.36 observed for the 2-h expressed enzyme (Table II); the apparent intensity ratio of the two mass peaks 2912.36/2913.36 was ~2.5:1 (Fig. 1A). For comparison, the tryptic peptide of the Asn376 right-arrow Asp mutant form revealed only a single monoisotopic peak of m/z 2913.36 (Fig. 1B). The tryptic peptide of Delta N-(1-102)/Delta C-(429-452)-hPAH truncated form expressed for 24 h at 28 °C gave a similar result as the wt-hPAH 24 h (data not shown). However, in this case also the 20-residue peptide (Phe131-Lys150) containing Asn133, with a single monoisotopic peak at m/z 2193.06 in the 2-h expressed enzyme (Table II), revealed on 24-h expression an additional peak at m/z 2194.07; the apparent intensity ratio of the two mass peaks 2193.06 was ~14.1:1 (Fig. 1C). For comparison, the tryptic peptide of the Asn133 right-arrow Asp mutant form revealed only one monoisotopic peak at m/z 2194.07 (Fig. 1D).


                              
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Table II
Asparagine containing tryptic peptides of recombinant wt-hPAH expressed for 2 h at 28 °C identified by MALDI-TOF mass spectrometry


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Fig. 1.   MALDI-TOF mass spectra of the tryptic peptides Thr372-Lys396 and Phe131-Lys150 of wt-hPAH containing labile Asn residues. The mass spectra were isotopically deconvoluted, and only the monoisotopic mass peaks ([M + H+]) are shown. A, the peptide Thr372-Lys396 from wt-hPAH expressed for 24 h at 28 °C; B, the same peptide from the Asn376 right-arrow Asp mutant form. C, the peptide Phe131-Lys150 from Delta N-(1-102)/Delta C-(429-452)-hPAH expressed for 24 h at 28 °C; D, the same peptide from the Asn133 right-arrow Asp mutant form.

Steady-state Kinetic Properties of Tetrameric Wild-type and Asn right-arrow Asp Mutant Forms of hPAH-- For tetrameric wt-hPAH it has been shown (4) that partial but multiple deamidated forms of hPAH change the catalytic properties of the enzyme. In the present study (Fig. 2 and Table III) the catalytic efficiency (kcat/[S]0.5) of the multiple deamidated wt-hPAH (24-h induction at 28 °C) was found to be about 3-fold higher than for the nondeamidated enzyme (2-h induction), i.e. 1.83 versus 0.72 µM-1·min-1, due to an increase (by 55%) in the Vmax and a reduction (by 46%) in the [S]0.5(L-Phe) value. Some of the Asn right-arrow Asp mutant tetramers (2-h induction) resulted in changes in the steady-state kinetic parameters comparable with those observed as a result of the deamidations of wt-hPAH (24-h induction), notably the N32D mutation in the regulatory domain and the mutations N167D and N376D in the catalytic domain (Table III). The largest increase (kcat/[S]0.5 = 1.58) was observed for the N376D mutant tetramer which revealed an increase (by 69%) in Vmax and a reduction (by 33%) in the [S]0.5(L-Phe) value compared with the wild-type control (2-h induction) (Table III). By contrast, a considerable reduction in catalytic efficiency was observed for the N207D, N223D, and N426D mutant tetramers, whereas no significant change was detected for the N393D mutant form (Table III), which revealed both reduced Vmax and [S]0.5 value for L-Phe.


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Fig. 2.   The effect of L-Phe concentration on the catalytic activity of the double mutant form N32D/N376D-hPAH. For assay conditions of the isolated tetrameric enzyme preparation and nonlinear regression analysis, see "Experimental Procedures." A, N32D/N376D-hPAH; B, wt-hPAH (2-h induction); C, wt-hPAH (24-h induction). In all graphs the experimental () and fitted (open circle ) data are shown. The kinetic constants are summarized in Table III.


                              
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Table III
Steady-state kinetic properties of tetrameric wild-type and mutant forms of hPAH
The wt-hPAH was isolated after an induction period of 2 and 24 h, whereas the Asn right-arrow Asp mutant forms were isolated after 2 h induction with IPTG.

An increased positive kinetic cooperativity of L-Phe binding as well as a substrate inhibition are also characteristic properties of multiple deamidated (24-h induction at 28 °C) wt-hPAH (4). The N32D, N167D, N376D, N401D, and N32D/N376D mutant tetramers revealed all the same positive cooperativity (1.8 < h < 2.0) as the multiple deamidated wt-hPAH as well as a substrate inhibition at L-Phe concentrations >1 mM (Table III). By contrast, the other Asn right-arrow Asp mutant forms did not reveal any substrate inhibition within the selected concentration range of L-Phe (<= 4 mM). For the N133D and N426D mutant forms a positive cooperativity (1.4 h < 1.6) comparable with wt-hPAH (2-h induction) (h = 1.5) was observed, whereas no significant cooperativity (0.8 < h < 1.2) was found for the N207D, N223D, and N393D mutant tetramers.

A slightly reduced affinity for the natural pterin cofactor has been observed for multiple deamidated hPAH when compared with the nondeamidated enzyme (4), and in the present study a 29% increase in the Km (H4biopterin) value was observed for the wt-hPAH (24-h induction). The N167D, N223D, N376D, and N401D mutant forms also demonstrated a significant increase in the Km value for the natural pterin cofactor, i.e. by 19-39%, compared with wt-hPAH (2-h induction) (Table III). By contrast, the N133D and N426D mutants revealed an affinity for the pterin cofactor comparable with wt-hPAH (2-h induction), whereas a reduction in the Km value by 74 and 35% was observed for the N207D and N393D mutants, respectively (Table III).

Effect of Preincubation with L-Phe on the Catalytic Activity-- A characteristic property of tetrameric wt-hPAH is also that preincubation with L-Phe resulted in an activation of the enzyme catalytic activity, and that the measured fold activation varied with the time of preincubation (4) due to a relatively slow substrate-induced hysteretic conformational transition (29). To study the effect of the Asn right-arrow Asp mutations on the activation process, the catalytic activities of both nondeamidated wt-hPAH and the mutant tetramers (2-h induction) were measured in the presence and absence of preincubation with 1 mM L-Phe. Table III shows that a 5-7-fold activation was observed for the N133D, N376D, N401D, and N426D mutant tetramers, which is in the same range of activation as observed for the 24-h-induced multiple deamidated wt-hPAH (5.8-fold) (Table III). The N167D, N223D, and N393D mutant tetramers revealed a lower degree of activation (i.e. 2.7-3.8-fold) than the wt-hPAH (2-h induction) (4.9-fold), whereas no L-Phe activation was observed for the N207D mutant form (Table III). Interestingly, the Asn32 right-arrow Asp mutant tetramer revealed the same degree of activation as the wt-hPAH (2-h induction), whereas an increased basal catalytic activity with almost no further stimulation by preincubation with Phe was observed for the Gly33 right-arrow Ala/Val mutations (Table III).

Limited Proteolysis by Trypsin-- Limited proteolysis by trypsin has been shown to represent a sensitive conformational probe in wt-hPAH (4) and some of its disease-associated mutant forms (26). That nondeamidated hPAH (2-h induction) was more resistant to limited proteolysis by trypsin than multiple deamidated hPAH (24-h induction) (4) was confirmed in the present time course studies, with about 85 and 52% of the full-length protomer recovered for the nondeamidated and multiple deamidated forms of wt-hPAH, respectively, after incubation for 15 min at a trypsin:protein ratio of 1:200 (by mass) (data not shown). Moreover, 6 of 8 Asn right-arrow Asp mutant tetramers (i.e. N167D, N207D, N223D, N376D, N393D, and N401D) revealed a variable but increased susceptibility to limited proteolysis when compared with the nondeamidated wild-type enzyme (2-h induction). The N207D mutant form was most susceptible to degradation, because only 19% full-length protomer was recovered after 15 min. By contrast, the N133D and N426D mutants gave a similar rate of proteolysis as the wt-hPAH (2-h induction) (data not shown).

Phosphorylation of hPAH-- In wt-hPAH Ser16 was a substrate for the catalytic subunit of PKA, and its phosphorylation sensitized the enzyme toward activation by L-Phe (30). Moreover, the rate of this phosphorylation was conformation-sensitive and was stimulated by L-Phe binding, whereas BH4 acted as a negative effector (30). In this study, we have further observed that the initial rate of phosphate incorporation was about 20% higher (p < 0.003) for the multiple deamidated enzyme (24-h induction) than for the nondeamidated enzyme (2-h induction) hPAH (Fig. 3). The N32D mutant form (2-h induction) also resulted in an enhanced rate of phosphorylation by PKA (Fig. 3), comparable (p < 0.2) to the deamidated wt-hPAH (24-h induction), whereas the G33A/G33V mutant forms (2-h induction) and the G33A (24-h induction) were phosphorylated with a similar initial rate as the nondeamidated wt (2-h expression) enzyme (Fig. 3).


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Fig. 3.   The effect of deamidation of Asn32 on the initial rate of phosphorylation of hPAH at Ser16 by PKA c-subunit. A, time course of phosphate incorporation of the deamidated wt-hPAH (24-h induction). The data points correspond to the average experimental values for the incorporation of 32Pi (n = 3). The initial rate of phosphorylation was calculated by the 15 individual data points obtained within the first 2.5 min, and the initial rate v0 (tangent to the curve at zero time) was calculated by nonlinear regression analysis. B, the bars with the mean ± S.E. represent the initial rates for the phosphorylation of deamidated and nondeamidated hPAH. Analysis of variance of the initial rate means indicated a statistical significant difference between the wt-hPAH (24 h) versus wt-hPAH (2 h) (p < 0.003) and the N32D (2 h) versus wt-hPAH (2 h) (p < 0.005). There was no statistical significant difference between the wt-hPAH (2 h) versus G33A (2 h), G33V (2 h), or G33A (24 h) (p < 0.6) nor between the wt-hPAH (24 h) versus N32D (2 h) (p < 0.2).

Reversed-phase Chromatography of Tryptic Peptides-- Together with Asn28 and Asn30, the very labile Asn32 is part of a cluster of Asn residues in the 28-residue N-terminal tryptic peptide (Leu15-Lys42), which also includes Ser16. Upon its phosphorylation with PKA and tryptic proteolysis, the 32P-labeled peptides were resolved by reversed-phase chromatography into some minor phosphopeptides and a major component (tR ~34 min), which revealed a time-dependent shift to a more hydrophilic position (tR ~31 min) when incubated at 37 °C (1). When the phosphopeptides resulting from tryptic proteolysis of the N32D and G33A/G33V mutant enzymes were subjected to reversed-phase chromatography, the major peptide of N32D revealed the expected retention time of tR ~31 min, while that of G33A/G33V mutant derived peptide eluted with a tR ~36 min, and none of them shifted on further incubation at pH 7 and 37 °C (Fig. 4) (results not shown for G33V). The minor components in the chromatograms can be explained as the result of alternative tryptic cleavage sites (1).


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Fig. 4.   Reversed-phase chromatography of 32P-labeled tryptic phosphopeptides of Asn32 right-arrow Asp and Gly33 right-arrow Ala mutant forms of hPAH. The full-length mutant tetramers were phosphorylated at Ser16 by PKA and digested with trypsin. The phosphopeptides were subjected to reversed-phase chromatography at time 0 () and after 32 h of incubation (- - -) in the phosphorylation medium at pH 7 and 37 °C. 250-µl fractions were collected every 15 s, followed by scintillation counting and analysis of the data by the PeakFit software program (SPSS Inc., Chicago). The elution pattern of the phosphopeptides revealed a main peak at tR ~31 min for the N32D and at tR ~36 min for the G33A mutant form. The heterogeneity of the eluted phosphopeptides may be related to alternative cleavage sites for tryptic proteolysis (1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The microheterogeneity of recombinant wt-hPAH expressed in E. coli observed on isoelectric focusing and two-dimensional electrophoresis has been shown to be the result of multiple nonenzymatic deamidations of Asn residues (1, 4). In general, such protein deamidations have been related to their aging and turnover in vivo (10, 31), which is also the case for hPAH. First, on expression of recombinant hPAH in E. coli, this post-translational modification was found to increase progressively with increasing induction time with IPTG and not to be related to the purification or storage of the enzyme. Second, highly deamidated forms of the enzyme are more susceptible to limited proteolysis by trypsin as compared with the nondeamidated form (4). Finally, PAH purified from human and monkey liver has also been reported to consist of multiple molecular forms on two-dimensional electrophoresis (2), which suggests that nonenzymatic deamidation of Asn residues plays a role in the regulation of the catalytic activity and the cellular turnover of PAH in the hepatocytes in vivo.

Asn Residues Undergoing Deamidation in Recombinant hPAH-- Based on a recently developed computer algorithm (10, 16), several candidate Asn residues have been predicted in the regulatory and catalytic domains of hPAH (17). The deamidation of the predicted residues in the autoregulatory sequence of the N-terminal regulatory domain (Asn28, Asn30, and Asn32) has been confirmed recently (1) by MALDI-TOF mass spectrometry, with Asn32 as the most labile residue. This deamidation has been studied further in the present study by reversed-phase chromatography of the phosphorylated (32P-labeled) N-terminal tryptic peptide (Leu15-Lys42) of the mutant N32D and G33A/G33V enzymes. The phosphopeptide of the N32D mutant form was eluted with the same retention time as the deamidated peptide of wt-hPAH incubated at 37 °C (main peak at tR ~31 min) (Fig. 4) (1). By contrast, the phosphopeptides of the G33A/G33V mutant enzymes were eluted with a higher retention time (tR ~36 min) (Fig. 4) comparable with that observed for the peptide of nondeamidated wt-hPAH (main peak at tR ~34 min) (1); the small difference in retention time may be explained by the substitution of a Gly with a larger aliphatic residue. Thus, as expected from studies on model peptides (10), the Gly33 right-arrow Ala/Val mutations stabilize Asn32 (see also Fig. 3).

On the basis of the amino acid sequence and higher order structure, the mentioned computer algorithm (16, 32) estimated a very low deamidation coefficient (CD = 5.5) for Asn376, indicating that it is one of the most labile Asn residues in the catalytic domain (Table IV). This prediction was verified by MALDI-TOF mass spectrometry and site-directed mutagenesis. Thus, the tryptic peptide Thr373-Lys392 of the wt-hPAH (24-h induction) was resolved into two monoisotopic ([M + H+]) peaks corresponding to nondeamidated (Asn) forms (Table II) and deamidated (Asp/iso-Asp) forms (Fig. 1A), whereas the N376D mutant revealed only one monoisotopic peak corresponding to an Asp residue in this 25-residue peptide (Fig. 1B). By contrast, two alternative deamidation coefficients were estimated for Asn133 on the basis of the three-dimensional structure of the Delta N-(1-117)-hPAH (CD = 4.1) and the Delta N-(1-102)/Delta C-(429-452)-hPAH (CD = 73) truncated forms (Table IV). The different CD values probably reflect an effect of the regulatory and tetramerization domains on the stability of Asn133. In the full-length wt-hPAH the native three-dimensional structure may contribute to additional structural constraints that protect against deamidation of Asn133. Thus, only one monoisotopic peak, corresponding to an asparagine residue at position 133, was found in the Phe131-Lys150 tryptic peptide for the wt-hPAH (24 h), and only a minor deamidated form was recovered in the same peptide from the Delta N-(1-102)/Delta C-(429-452)-hPAH truncated from (Fig. 1C). These results, together with the deamidation coefficient estimated for this residue in the phosphorylated form of rPAH-Delta C22 (17), show that Asn133 represents the second labile Asn in the catalytic domain of hPAH.


                              
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Table IV
An overview of the nearest neighbor amino acids, secondary structure, and three-dimensional structural interactions of the Asn residues in the catalytic domain of hPAH and their predicted deamidation rates
t1/2 represents the first-order deamidation half-times predicted from the primary amino acid sequence (10, 17), and CD is the deamidation coefficient, an estimate of the relative rates of deamidation based on both the nearest neighbor amino acids and the three-dimensional structure of Delta N117-hPAH (Protein Data Bank code 2PAH) (17).

Site-directed Mutagenesis-- Mutagenesis (Asn right-arrow Asp) of the three labile Asn residues Asn32, Asn376, and Asn133 has provided useful models to study the contribution of each single Asn deamidation to the changed functional properties that occur in wt-hPAH on long term expression in E. coli. It should be noted, however, that the introduction of a negative charge by Asn right-arrow Asp mutagenesis does not fully account for the effect of deamidation of wt-hPAH in which iso-Asp might also be formed. The generation of an iso-Asp residue, which is a regular product of the deamidation reaction, also results in a conformational change because it adds an extra carbon to the polypeptide backbone (33). Thus, the formation of iso-Asp has been shown recently for the deamidation of alternative Asn residues in wt-hPAH including Asn32 (1), as expected from the Asn32-Gly33 sequence and the high crystallographic B factor observed for this region in the crystal structure of rPAH (34).

The N32D mutation revealed catalytic properties similar to that of multiple deamidated wt-hPAH (24 h expression), i.e. an increased catalytic efficiency and an increased positive cooperativity of L-Phe binding and substrate inhibition (Table III), whereas substitution of Gly33 with Ala/Val residues, which prevents the deamidation of Asn32, resulted in enzyme preparations with kinetic properties similar to that observed for wt-hPAH (2-h expression). Moreover, the N32D mutant form and the wt-hPAH (24 h) showed similar increased rates of phosphorylation of Ser16 by PKA when compared with the G33A and G33V mutant forms and the wt-hPAH (2 h) (Fig. 3). Deamidation of Asn32 into an Asp/iso-Asp residue adds a negative charge to the side chain that results in an electrostatic repulsion notably from Asp84 (1) and results in a conformational change, as determined by its effect on the rate of phosphorylation of Ser16 by PKA. Deamidation of Asn32 seems to be the only determinant for this effect, because the partial deamidation of the other asparagine residues, which occur in the G33A mutant form on 24-h induction, did not affect the initial rate of phosphorylation (Fig. 3).

In the catalytic domain, Asn376 was shown to represent the most labile Asn residue. In the crystal structure it is located at the beginning of a loop (designated the 380-loop) that represents one limiting side of the active site crevice structure (35) (Table IV; Fig. 5). On binding of the pterin cofactor (H4biopterin) at the active site a conformational change, including a movement of this loop, has been observed for a double truncated form ("catalytic domain") of hPAH (35). On this background, it was not unexpected to find that the steady-state kinetic parameters of the N376D mutant tetramer were comparable with those observed for multiple but partially deamidated wt-hPAH (24-h induction) (Table III). Its Vmax was slightly higher than that of the multiple deamidated wt-hPAH and, in addition, a reduction by 33% in the [S]0.5(L-Phe) value and an increase (by 39%) in the Km(H4biopterin) was observed as compared with 46 and 29%, respectively, for the multiple deamidated (24-h induction) wt-hPAH (Table III). The N376D mutant form also revealed an increase in the fold activation on preincubation with L-Phe when compared with the wt-hPAH (2-h induction) (Table III), similar to that observed for the multiple deamidated hPAH (24-h induction). It has been proposed previously (34) that the autoregulatory sequence of the N-terminal regulatory domain partly blocks the access of substrate to the active site in the catalytic domain. On preincubation with L-Phe, there is a global conformational change in the enzyme (29), including a possible repositioning of the N-terminal "arm" relative to the active site (34), and an activation of the enzyme. A hinge region centered at Gly33 has been proposed to be involved in this repositioning (34), on the basis of a high crystallographic B-factor in this region of the rat enzyme, compatible with a high mobility. Although a movement at this hinge region is probably involved in the regulation of the access of substrate to the active site, deamidation of Asn32 does not seem to be involved in the increased L-Phe induced activation observed for the deamidated wt-hPAH (24 h), because the N32D mutant form revealed a similar degree of substrate activation as the wt-hPAH (2 h) (Table III). By contrast, in the Gly33 right-arrow Ala/Val mutant forms the orientation of this arm seems to generate a more open entrance for the substrate to the active site, and thus explains their higher basal catalytic activity and reduced activation by L-Phe preincubation. In the catalytic domain, deamidation of Asn376 significantly contributes to the increased substrate activation, as seen from the data (Table III) obtained for the N376D and the N32D/N376D mutant forms, when compared with the wt-hPAH (2 h). The second labile Asn residue in the catalytic domain, Asn133, is located in a long (18 residue) loop structure between the Calpha 1- and Calpha 2a-helix (Table IV; Fig. 5), and a minor partial deamidation of Asn133 was observed in enzyme preparations recovered after 24-h expression. However, the N133D mutant tetramer revealed steady-state kinetic properties comparable with that observed for the nondeamidated wt-hPAH (2 h) (Table III). These results, together with the mass spectrometry data, indicate that deamidation of Asn133 is unlikely to contribute to the kinetic differences observed between the 2- and the 24-h expressed enzyme.


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Fig. 5.   Interactions of the asparagine residues in the catalytic domain with the neighbor amino acids in the hPAH structure. The figure was based on the crystal structure of the ligand-free dimeric Delta N-(1-102)/Delta C-(428-452)-hPAH form (Protein Data Bank code 1PAH) containing the residues 117-424. Rotated enlargements (A-D) are shown of the corresponding regions containing structurally important asparagine residues. The figures were made using the program Web Lab Viewer.

Considering all the data discussed above, the double mutant form N32D/N376D represents so far the best model enzyme which mimics the highly deamidated wt-hPAH (24 h). It is also a protein preparation with less microheterogeneity and may thus represent an interesting molecular form in the current efforts to crystallize and solve the structure of the full-length tetrameric enzyme. Thus, nonenzymatic deamidation of Asn residues introduces a time-dependent microheterogeneity that may be related to the reported time-dependent loss of diffraction power of the hPAH crystals (36).

Structural and Functional Considerations of the Stable Asn Residues in the Catalytic Domain-- Although the six stable Asn residues observed in the catalytic domain of hPAH are all located on the surface (Table IV), their nearest neighbor amino acids and the structural constraints in the polypeptide backbone represents the protection against their deamidation (17) (Table IV). Asn167, Asn207, Asn393, Asn401, and Asn426 have rather long predicted half-times for deamidation (between 10 and 70 days) based on their nearest neighbor amino acids (10, 17), and they are in addition stabilized by hydrogen bonds and/or by their location in alpha -helical structures (Table IV), which results in high deamidation coefficients (40 < CD < 2100) (Table IV) (17). This computational prediction of stable Asn residues was confirmed by MALDI-TOF mass spectrometry of the corresponding tryptic peptides from the 24-h expressed wt-hPAH with single monoisotopic mass peaks, corresponding to fully amidated tryptic peptides as seen for the 2-h expressed enzyme (Table II). Moreover, five residues (Asn167, Asn207, Asn223, Asn393, and Asn426) are involved in at least one stabilizing intrasubunit hydrogen bond (Fig. 5). On Asn right-arrow Asp mutation of these residues, their intrasubunit and solvent contacts change more than for the nonhydrogen-bonded residues (Asn133, Asn376, and Asn401).

Asn167 is located just after the Calpha 2b-helix and is stabilized by a single hydrogen bond to the carbonyl oxygen of Asp163 (Table IV; Fig. 5). Interestingly, in the N167D mutant tetramer the repulsive electrostatic interaction between the negatively charged carboxyl groups of Asp167 and Asp163 resulted in similar kinetic properties of the mutant form as the wt-hPAH (24-h induction) (Table III). The importance of the conserved Asn residue at this position is further supported by the finding of an Asn167 right-arrow Ile mutation in a PKU patient (37). Moreover, Asn207 and Asn223 also seem to have a structural role in hPAH. The substitution of Asn207 and Asn223 by Asp residues resulted in tetrameric proteins with kinetic properties very different from both the nondeamidated and deamidated wt-hPAH, including a reduced catalytic efficiency (Table III). Asn223 is located in a loop between the Calpha 4 and Calpha 5 helices but is stabilized by hydrogen bonding to two residues (Table IV; Fig. 5). The same is true for Asn393, located in a long loop structure between the Calpha 11 and Calpha 12 helices, and is stabilized by two hydrogen bonds to Ser298 (Table IV; Fig. 5). The N393D mutant tetramer revealed a low positive kinetic cooperativity of L-Phe binding and a low degree of substrate activation, as compared with the wt-hPAH (2 h), and thus support a stabilizing structural/functional role of this residue as well. With respect to Asn401, this residue is located in the middle of the Calpha 12-helix (38) and was therefore not considered to be a candidate for deamidation, although the N401D mutant enzyme revealed kinetic properties comparable with the highly deamidated wt-hPAH (Table III). Finally, it should be mentioned that Asn426 is a highly conserved residue within mammalian PAHs, and although hydrogen-bonded to the Phe410 main chain, it is part of a short flexible hinge region (Asp425-Gln429) (36) just in front of Calpha 14-helix (38), the beginning of the tetramerization motif (Table IV). This short hinge region (Asp425-Gln429) has been assigned a function in the tetramerization of dimers and the cooperative binding of substrate to tetrameric wt-hPAH (23). Interestingly, in contrast to the Thr427 right-arrow Pro substitution (23), the Asn426 right-arrow Asp mutation did not interfere with the tetramer left-right-arrow dimer equilibrium characteristic of wt-hPAH nor with the limited tryptic proteolysis (data not shown) or the cooperativity for L-Phe of the tetrameric enzyme (Table III). By MALDI-TOF mass spectrometry of the tryptic peptides from wt-hPAH, it was also possible to recover the peptide containing Asn438 (Table II) which did not show any evidence of deamidation in the 24-h expressed enzyme, as predicted from the estimated deamidation coefficient (CD = 46) (17). Asn438 is located on the terminal alpha -helix that is part of the tetramerization domain, and interactions with the other amino acids in the helical structure are expected to protect it from undergoing a deamidation reaction (14).

The Enzyme Phenotype of the Disease-associated Mutations Asn167 right-arrow Ile and Asn207 right-arrow Asp-- More than 400 mutations associated with PKU/hyperphenylalanineemia have been identified so far in the PAH gene (39) (data.mch.mcgill.ca/pahdb_new), but only three of them have been reported in the codons for Asn residues, i.e. N167I, N207D, and N207S. The N167I mutation has been found in a few PKU patients in Wales, Scotland, and Belgium (37), but so far no information has been reported on their metabolic and enzymatic phenotypes. Compared with wt-hPAH (2-h induction at 28 °C), the N167I mutation revealed an almost 2-fold higher catalytic efficiency and a higher Hill coefficient for the L-Phe binding, although the apparent Vmax was reduced by about 50% (Table III). In addition, the Km value for the pterin cofactor was decreased (Table III). Thus, the kinetic properties of the recombinant N167I tetramer do not explain why the mutation is associated with PKU. However, some PKU-related mutant forms are characterized by their reduced cellular stability (20, 26, 40), and degradation of misfolded mutant proteins has been proposed as a general mechanism whereby a missense mutation leads to PKU/hyperphenylalanineemia (20, 26, 40). The N167I mutant gave a low recovery of the tetrameric form on size-exclusion chromatography after cleavage of the fusion protein (data not shown) due to aggregation, and the steady-state kinetics were therefore determined using the tetrameric fusion protein. An isoleucine at this position may destabilize the secondary structure (Fig. 5), and the presence of a hydrophobic residue on the outside of the alpha -helix, accessible to the solvent, may explain the tendency of the N167I mutant form to aggregate. Because codon 167 is located at the end of exon 5 in the hPAH gene, one should also consider the possibility that the mutation may affect the splicing of this exon. The N207D mutation has been identified in a Korean PKU patient as a compound heterozygote with the mutant allele Y325X (41), but no information has yet been reported on the metabolic and enzymatic phenotype of this heteroallelic genotype. The recombinant N207D mutant tetramer revealed a decreased Vmax value as well as a decreased affinity for L-Phe and an increased affinity for the pterin cofactor relative to wt-hPAH, but it did not show any kinetic cooperativity of L-Phe binding and no activation by L-Phe preincubation. Furthermore, the dramatic effect shown on tryptic proteolysis and temperature stability by the isolated tetrameric mutant enzyme indicates that the mutant protein is in a rather open conformational state and thus more susceptible to intracellular proteolysis than the wild-type hPAH. Thus, the expected enzymatic and metabolic phenotype of the N207D/Y325X alleles is a classic PKU. The Asn207 is located in the middle of the Calpha 4-helix of hPAH and is a highly conserved residue in PAH of different species, also suggesting an important structural role. It is stabilized by a hydrogen bond between Asn207Ndelta 2 and Ala202 carbonyl oxygen (3.13 Å) (located in the loop between Calpha 3-helix and Calpha 4-helix) and an electrostatic attraction to Tyr198 carbonyl oxygen (3.6 Å) (located in the end of Calpha 3-helix) (Fig. 5). The negative charge on the mutant Asp residue is likely to induce an electrostatic repulsion with both the carbonyl groups of Ala202 and Tyr198 (Fig. 5) and therefore disrupt the tertiary structure in this region by moving the alpha -helices away from each other.

Concluding Remarks-- Post-translational modifications of proteins are important in the regulation of many cellular processes, providing a way to specifically change the structure and function of target proteins. Although phosphorylation and acetylation have received the most attention, nonenzymatic deamidation of labile Asn residues is also a frequently occurring post-translational modification in proteins (42). The reaction mechanism of this hydrolytic reaction (6) and the main factors that determine the wide range of rate constants observed in individual proteins (and peptides) (42) are also well characterized. Recently, a computer algorithm was developed for the precise prediction of these rate constants, based on the contribution of the primary structure (notably the nearest neighbor amino acids in the n + 1 position) and the three-dimensional structure (16, 32). Our recent (1) and present studies on the multiple Asn deamidations in hPAH represent another example of the high predictive value of this algorithm. Moreover, it is also well established that the deamidations of Asn residues in target proteins occur in vivo at rates that often exceed those observed in vitro (42), as first shown for the multiple deamidations in the mitochondrial protein cytochrome c (31), due to the effects of the physiological temperature (37 °C) and solution properties (7, 42). Much less information is, however, available on the effects of deamidation on the structure and function of the target proteins. The deamidation of Asn residues results in two products, Asp and iso-Asp (9, 33), which have the same effect on the net charge of the protein. Because iso-Asp also changes the peptide backbone (33), it is, however, not straightforward to predict the overall structural effect (structural isomerization) of protein deamidation(s). On the other hand, their functional effects can be tested more easily. Thus, it has been reported that deamidation(s) of Asn residues in proteins can both markedly reduce (15) or increase (Refs. 1 and 4 and the present study) their biological activity and may also represent a "molecular timer" in the turnover of relatively long lived proteins (10). The most dramatic biological effect of protein deamidation was recently described for the anti-apoptotic protein Bcl-XL (43). Thus, on deamidation of Asn52 and Asn66 in a surface-exposed loop structure, the anti-apoptotic activity is lost by changing its interaction with target proteins (BH3 domain-only proteins). Equally interesting was the finding that the deamidations were observed to be suppressed by the interaction of Bcl-XL with a specific retinoblastoma protein. Thus, specific protein-protein interactions may represent a third structural factor in determining the rate of deamidation of Asn residues in target proteins in vivo.

    ACKNOWLEDGEMENTS

We thank Ali Sepulveda Muñoz for expert technical assistance in preparation of bacterial extracts. The collaboration with Dr. Dietmar Waidelich in the MALDI-TOF analysis is greatly appreciated.

    FOOTNOTES

* This work was supported in part by Fundação para a Ciência e Tecnologia, Portugal, Grants SFRH/BD/1100/2000, the Research Council of Norway, and the University of Bergen.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 and Molecular Biology, University of Bergen, Årstadveien 19, N-5009 Bergen, Norway. Tel.: 47 55586428; Fax: 47 55586400;. E-mail: torgeir.flatmark@ibmb.uib.no.

Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212180200

    ABBREVIATIONS

The abbreviations used are: PAH, phenylalanine hydroxylase; hPAH, human phenylalanine hydroxylase; rPAH, rat phenylalanine hydroxylase; H4biopterin, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin; MALDI-TOF, matrix-assisted desorption/ionization time of flight; IPTG, isopropyl-beta -D-galactoside; L-Phe, L-phenylalanine; MBP, maltose-binding protein; wt, wild-type; PKA, cAMP-dependent protein kinase; PKU, phenylketonuria.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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