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INTRODUCTION |
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
-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
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.
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EXPERIMENTAL PROCEDURES |
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.
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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-
-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 ([
-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.
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RESULTS |
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
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
N-(1-102)/
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
Asp mutant form revealed
only a single monoisotopic peak of m/z 2913.36 (Fig. 1B). The tryptic peptide of
N-(1-102)/
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
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 Asp mutant form. C,
the peptide Phe131-Lys150 from
N-(1-102)/ C-(429-452)-hPAH expressed for 24 h at 28 °C;
D, the same peptide from the Asn133 Asp
mutant form.
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Steady-state Kinetic Properties of Tetrameric Wild-type and Asn
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
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 ( ) 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 Asp mutant forms were isolated after 2 h
induction with IPTG.
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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
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
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
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
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
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).
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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 Asp and
Gly33 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).
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DISCUSSION |
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
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
N-(1-117)-hPAH (CD = 4.1) and the
N-(1-102)/
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
N-(1-102)/
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-
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 N117-hPAH
(Protein Data Bank code 2PAH) (17).
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Site-directed Mutagenesis--
Mutagenesis (Asn
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
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
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 C
1- and C
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 N-(1-102)/ 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.
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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
-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
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 C
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
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 C
4 and
C
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 C
11 and C
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 C
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
C
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
Pro
substitution (23), the Asn426
Asp mutation did not
interfere with the tetramer
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
-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
Ile and Asn207
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
-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 C
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 Asn207N
2 and
Ala202 carbonyl oxygen (3.13 Å) (located in the loop
between C
3-helix and C
4-helix) and an electrostatic attraction to
Tyr198 carbonyl oxygen (3.6 Å) (located in the end of
C
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
-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.