Báo cáo khoa học: Studies on the regulatory properties of the pterin cofactor and dopamine bound at the active site of human phenylalanine hydroxylase pptx

Studies on the regulatory properties of the pterin cofactor
and dopamine bound at the active site of human phenylalanine
hydroxylase
Therese Solstad
1
, Anne J. Stokka
1
, Ole A. Andersen
2
and Torgeir Flatmark
1
1
Department of Biochemistry and Molecular Biology, University of Bergen, Norway;
2
Department of Chemistry,
University of Tromsø, Norway
The catalytic activity of phenylalanine hydroxylase (PAH,
phenylalanine 4-monooxygenase EC 1.14.16.1) is regulated
by three main mechanisms, i.e. substrate (
L
-phenylalanine,
L-Phe) activation, pterin cofactor inhibition and phos-
phorylation of a single serine (Ser16) residue. To address the
molecular basis for the inhibition by the natural cofactor
(6R)-
L
-erythro-5,6,7,8-tetrahydrobiopterin, its effects on the
recombinant tetrameric human enzyme (wt-hPAH) was
studied using three different conformational probes, i.e. the
limited proteolysis by trypsin, the reversible global con-
formational transition (hysteresis) triggered by L-Phe bind-

ing, as measured in real time by surface plasmon resonance
analysis, and the rate of phosphorylation of Ser16 by cAMP-
dependent protein kinase. Comparison of the inhibitory
properties of the natural cofactor with the available three-
dimensional crystal structure information on the ligand-free,
the binary and the ternary complexes, have provided
important clues concerning the molecular mechanism for
the negative modulatory effects. In the binary complex,
the binding of the cofactor at the active site results in the
formation of stabilizing hydrogen bonds between the
dihydroxypropyl side-chain and the carbonyl oxygen of
Ser23 in the autoregulatory sequence. L-Phe binding triggers
local as well as global conformational changes of the pro-
tomer resulting in a displacement of the cofactor bound at
the active site by 2.6 A
˚
(mean distance) in the direction of the
iron and Glu286 which causes a loss of the stabilizing
hydrogen bonds present in the binary complex and thereby a
complete reversal of the pterin cofactor as a negative effector.
The negative modulatory properties of the inhibitor dop-
amine, bound by bidentate coordination to the active site
iron, is explained by a similar molecular mechanism inclu-
ding its reversal by substrate binding. Although the pterin
cofactor and the substrate bind at distinctly different sites,
the local conformational changes imposed by their binding
at the active site have a mutual effect on their respective
binding affinities.
Keywords: tetrahydrobiopterin; dopamine; phosphoryl-
ation; surface plasmon resonance; regulation.

Mammalian phenylalanine hydroxylase (PAH, phenylala-
nine 4-monooxygenase, EC 1.14.16.1) catalyses the stereo-
specific hydroxylation of
L
-phenylalanine (L-Phe) to
tyrosine (L-Tyr) in the liver [1], kidney [2,3] and melano-
cytes [4], utilizing (6R)-
L
-erythro-5,6,7,8-tetrahydrobiop-
terin (H
4
biopterin) as the physiological electron donor. A
lack or dysfunction of this enzyme in humans is associated
with the autosomal recessive disease hyperphenylalanine-
mia/phenylketonuria [5] ( It
has been estimated that the liver contains a sufficiently high
level of PAH and pterin cofactor to remove all free L-Phe
from the blood within a few minutes if all enzyme molecules
are fully active [6]. However, early on it was recognized that
the activity of PAH is effectively controlled by several
mechanisms in order to maintain the phenylalanine and
tyrosine homeostasis in vivo despite great fluctuations in the
dietary intake of L-Phe and the overall rate of protein
catabolism. It became apparent that the short-term control
of rat liver PAH (rPAH) is kinetic, primarily through an
activation of the enzyme by L-Phe [7,8]. This activation is a
cooperative, reversible process involving all protomers of
the 200-kDa enzyme homotetramer [7,8]. rPAH is activated
several fold in vitro by preincubation with L-Phe as well as
by some amino acid analogues [9]. The recombinant human

enzyme (hPAH) has similar regulatory properties in vitro as
rPAH, i.e. the tetrameric form binds L-Phe with positive
cooperativity with a Hill coefficient (h) of 1.6–1.9, and
preincubation with substrate results in a sixfold to eightfold
activation of the enzyme [10]. In addition to its catalytic
function as an electron donor in the reduction of the
Correspondence to T. Flatmark, Department of Biochemistry and
Molecular Biology, University of Bergen, A
˚
rstadveien 19,
N-5009 Bergen, Norway.
Fax: + 47 55586400, Tel.: + 47 55586428,
E-mail: torgeir.fl
Abbreviations: hPAH, human phenylalanine hydroxylase; rPAH,
rat phenylalanine hydroxylase; h, Hill coefficient; H
2
biopterin,
(6R)-
L
-erythro-7,8-dihydrobiopterin; H
4
biopterin, (6R)-
L
-erythro-
5,6,7,8-tetrahydrobiopterin; H
4
6-methyl-pterin, 6-methyl-5,6,7,
8-tetrahydropterin; IPTG, isopropyl-thio-b-
D
-galactoside;

L-Phe,
L
-phenylalanine; MBP, maltose binding protein;
PKA, cAMP dependent protein kinase; wt, wild-type.
Enzyme: phenylalanine 4-monooxygenase or phenylalanine
hydroxylase (EC 1.14.16.1).
(Received 22 October 2002, revised 15 January 2003,
accepted 20 January 2003)
Eur. J. Biochem. 270, 981–990 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03471.x
catalytic iron and activation of dioxygen, the natural pterin
cofactor H
4
biopterin inhibits the substrate activation of
rPAHbyformingabinaryenzymeÆH
4
biopterin complex
[7,11]. The activity of rPAH and human phenylalanine
hydroxylase (hPAH) is also regulated by post-translational
mechanisms, notably phosphorylation of Ser16 by cAMP-
dependent protein kinase (PKA), which sensitizes the
enzyme for activation by L-Phe [12,13]. Whereas the
binding of substrate slightly increases the rate of phos-
phorylation of rPAH and hPAH by PKA, H
4
biopterin acts
as a negative effector on the same process [12,13]. Finally,
nonenzymatic deamidation of labile Asn residues in hPAH
during its expression in, e.g. Eschericia coli,hasmore
recently been shown to result in a threefold increase in its
catalytic efficiency [10].

The molecular basis for the inhibitory effects observed for
the natural pterin cofactor (H
4
biopterin/H
2
biopterin) has
been addressed in a series of studies on rPAH including
direct binding measurements [14], steady-state kinetic ana-
lysis [11] and modulation of Ser16 phosphorylation by PKA
[12]. The binding studies by Shiman and collaborators
[11,15] were interpreted to support a working model that
includes three types of binding sites for H
4
biopterin. The
sites were (a) a redox site, involved in the reduction of the
active site iron [Fe(III) fi Fe(II)], (b) a catalytic site,
involved in the activation of dioxygen and hydroxylation
of L-Phe, and (c) a regulatory site outside the active site,
responsible for its inhibitory properties. However, recent
crystal structure analyses of the ligand-free, the binary and
ternary complexes of hPAH [16–21], as well as the
complementary NMR-molecular modelling structural stud-
ies [22], have not been able to identify more than a single
cofactor binding site, i.e. the binding at the active site, with
two alternative orientations in the binary and ternary
complex [20,21]. Thus, the molecular basis for the regula-
tory (inhibitory) properties of H
4
biopterin is still a matter of
debate, both with respect to the domain localization of the

inhibitory binding site and the essential importance of its
dihydroxypropyl side-chain for inhibition. In the present
study, we address these questions as well as the related
regulatory properties of the catecholamine inhibitor dop-
amine, which is known to bind covalently (bidentate
coordination) to the active site iron [17] with high affinity,
employing recombinant tetrameric wt-hPAH, with the
three-dimensional crystal structural information on this
enzyme [16–21] as a reference.
Materials and methods
Materials
The restriction proteases enterokinase and factor Xa were
obtained from Invitrogen (The Netherlands) and Protein
Engineering Technology (ApS, Aarhus, Denmark), respect-
ively. The catalytic subunit of cAMP-dependent protein
kinase was purified to homogeneity from bovine heart and
was a generous gift from S. O. Døskeland, Department of
Anatomy and Cell Biology, University of Bergen. The
pterin cofactors (H
4
biopterin, H
2
biopterin and H
4
6-methyl-
pterin) were purchased from B. Schircks Laboratory
(Joana, Switzerland). Specific chemicals are mentioned in
the text elsewhere.
Expression and purification of recombinant hPAH
The pMAL expression system was used for the production

of the wild-type fusion proteins MBP-(D
4
K)
ek
-hPAH,
MBP-(IEGR)
Xa
-hPAH and its double truncated form
MBP-(IEGR)
Xa
-hPAH(Gly103-Gln428) with maltose
binding protein as the fusion partner [23]. Cells were grown
at 37 °C, and expression was induced at 28 °Cbythe
addition of 1 m
M
isopropyl-thio-b-
D
-galactoside (IPTG);
the cells were harvested after 2, 8 or 24 h of induction. Full-
length hPAH (residues 1–452) was obtained by enterokinase
(at D
4
K) or factor Xa (at IEGR) cleavage and
hPAH(Gly103–Gln428) by factor Xa cleavage, and the
tetrameric (full-length form) and dimeric form (the trun-
cated catalytic core enzyme) were isolated by size-exclusion
chromatography [23].
Protein measurements
The concentration of purified enzyme forms was measured
by the absorbance at 280 nm, using the absorption coeffi-

cient e
280
¼ 1.63 for the fusion protein MBP-(pep)-hPAH
and 1.0 for the isolated hPAH protein [23].
Protein phosphorylation
The tetrameric wt-hPAH was phosphorylated by PKA as
described [13]. The standard reaction mixture contained
15 m
M
Na-Hepes (pH 7.0), 0.1 m
M
ethylene glycol bis
(a-amino ether)-N,N,N¢,N¢-tetraacetic acid, 0.03 m
M
EDTA,
1m
M
dithiothreitol, 10 m
M
magnesium acetate, 60 l
M
[c-
32
P]-ATP (Amersham, UK), 25 n
M
of the catalytic
subunit of PKA and 4 l
M
of hPAH. The reaction was
performed at 30 °C, and at timed intervals, aliquots of the

reaction mixture were spotted on phosphocellulose strips
[24] to measure the amount of
32
P transferred to the
substrate. In control experiments, the active-site iron in
hPAH was prereduced by the noninhibitory [12] H
4
6-
methyl-pterin as described [14] prior to initiation of the
phosphorylation reaction in the presence of inhibitory
H
4
biopterin.
Limited proteolysis by trypsin
Limited tryptic proteolysis of tetrameric wt-hPAH was
performed at 25 °Cin20m
M
Na-Hepes, 200 m
M
NaCl at
pH 7; the ratio of trypsin to hPAH was 1 : 200 (m/m).
Aliquots of 3 lg hPAH were removed at timed intervals
and mixed with SDS buffer containing soybean trypsin
inhibitor. The protein was finally subjected to SDS/PAGE
(10%, w/v), stained with Coomassie Brilliant Blue, and the
gels were scanned using
DESKSCAN II
(Hewlett Packard Co)
and further analyzed by using the
PHORETIX

1
D
analysis
software from Nonlinear Dynamics Ltd [10].
Reversed-phase chromatography
Reversed-phase chromatography of tryptic peptides was
performed using a ConstaMetric Gradient System
(Laboratory Data Control, USA) and a 4.6 mm · 10 cm
Hypersil ODS C18 column (Hewlett Packard, USA)
fitted with a 2-cm guard column. Solvent A was 0.1%
982 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003
(w/v) triflouroacetic acid in water, and solvent B was
0.1% trifluoroacetic acid in 70% (v/v) acetonitrile.
A linear gradient of 5–100% solvent B at 1 mLÆmin
)1
for 60 min was used for the separation of peptides.
Absorbance was monitored at 214 nm using a Hewlett-
Packard model 1040A photodiode array HPLC detector.
Surface plasmon resonance analyses
The interactions between tetrameric wt-hPAH and its
substrates (L-Phe and H
4
biopterin/H
2
biopterin) were
studied by surface plasmon resonance (SPR) analysis
using the BiaCore X instrument (BiaCore AB, Uppsala,
Sweden). The enzyme, diluted to a final concentration of
0.23 mgÆmL
)1

in 10 m
M
sodium acetate buffer, pH 5.4, was
immobilized to the carboxymethylated dextran matrix of a
sensor chip (CM5 from BiaCore AB) by the amine coupling
procedure [25–27]. The double truncated dimeric form
hPAH(Gly103-Gln428) was immobilized to the reference
surface by the same procedure [27]. Seventy microlitres of
10 m
M
dithiothreitol in HBS running buffer (0.15
M
NaCl,
3m
M
EDTA, 0.005% surfactant P20 in 0.01
M
Hepes,
pH 7.4) was allowed to pass through both flow cells (in
series) which empirically decreased the time required to
reach a stable baseline [25]. Increasing concentrations of
L-Phe diluted in HBS buffer was injected over the immo-
bilized enzyme in the absence and presence of 100 l
M
H
4
biopterin or 500 l
M
H
2

biopterin. The reduced cofactor
(in 1 m
M
HCl) was kept on ice and the pH adjusted to 7.4
with an accuracy of 0.01 unit immediately before injection
[25]. All analyses were carried out at 25 °C and at a constant
flow of 5 lLÆmin
)1
. The sensorgrams were obtained as the
difference in SPR (DRU) response between the sample and
the reference cell, which corrects for any changes in the bulk
refractive index, together with the small DRU value
associated with the binding of the 165-Da substrate. The
time-dependent increase in RU (end point at 3 min) was
measured directly from the sensorgrams using the cursor
guided reading of the X- and Y-coordinates, and
was related to the calculated pmol of immobilized
enzyme by assuming that 1000 RU corresponds to 1 ng
protein boundÆmm
)2
[28], i.e. expressed as DRU/(pmol
subunitÆmm
)2
).
Structural studies
The recently reported three-dimensional crystal structures
of hPAH and rPAH [16–21] have provided the basis to
further explore the molecular mechanism by which the
pterin cofactor and dopamine function as negative effectors
and how substrate binding completely reverses these effects.

The coordinates for the structure of the binary and ternary
complexes of the catalytic core domain hPAH(Gly103-
Gln428), i.e. of hPAH-Fe(III)ÆH
2
biopterin (PDB id codes
1DMW and 1LRM) [18,20], of hPAH-Fe(II)ÆH
4
biopterin
(PDB id code 1J8U) [20] and hPAH-Fe(II)ÆH
4
biopterinÆ
3-(2-thienyl)-
L
-alanine (PDB id code 1KW0) [21], define the
position, orientation, conformation and hydrogen bonding
network of the pterin cofactor in the three structures.
Superpositions of the binary and ternary complexes of
hPAH onto the crystal structure of the ligand-free dimeric
rPAH (PDB id code 1PHZ) [19], which contains both the
regulatory and the catalytic domains (residues 1–429), were
performed to demonstrate the interactions of the dihydroxy-
propyl side-chain of the pterin cofactors with the N-terminal
autoregulatory sequence. Similarly, the coordinates for
the structure of the binary complex with dopamine, i.e.
hPAH-Fe(III)Ædopamine (PDB id code 5PAH) [17] define
the position and orientation of the inhibitor and the
interaction of its main-chain with the autoregulatory
sequence.
Results
Recombinant forms of hPAH were produced at high yields

as fusion proteins in E. coli using the pMAL expression
vector, and the cleaved tetrameric forms of wt-hPAH and
the dimeric truncated form hPAH(Gly103–Gln428) were
isolated by size-exclusion chromatography [23]. Aliquots
(10 mgÆmL
)1
)werestoredinliquidnitrogenandanew
aliquot was used for each individual experiment. The
conformational differences of tetrameric wt-hPAH resulting
from the binding of different ligands (H
4
biopterin, H
2
biop-
terin, dopamine and L-Phe) were studied using three
different conformational probes, i.e. their effects (a) on the
limited proteolysis by trypsin, (b) on the reversible con-
formational transition (hysteresis) triggered by substrate
binding, as followed in real time by surface plasmon
resonance (SPR) analyses, and (c) on the rate of phos-
phorylation of Ser16 by PKA.
Effect of ligands on the limited proteolysis
of recombinant wt-hPAH and its catalytic core
Limited proteolysis of rPAH by chymotrypsin has been
shown to be a sensitive conformational probe, and among
the observed ligand effects was an inhibition of proteolysis
by H
4
biopterin [29]. From Fig. 1A it is seen that at
saturating concentration (40 l

M
), H
4
biopterin has a similar
inhibitory effect on the limited proteolysis of tetrameric
wt-hPAH by trypsin, which cleaves after Lys/Arg residues
in a putative hinge region (residues 111–117, RDKKKNT)
connecting the regulatory domain with the catalytic domain
[30]. Moreover, the covalently bound inhibitor dopamine
(40 l
M
) is equally efficient in reducing the susceptibility
towards proteolysis. However, when 1 m
M
L-Phe was also
present during the incubation with trypsin, the inhibitory
effects of H
4
biopterin and dopamine were completely
prevented and the rate of proteolysis increased to approxi-
mately the same high level as observed in the presence of
substrate alone. By contrast, the dimeric catalytic core
enzyme hPAH(Gly103–Gln428) [16], lacking both the
N-terminal regulatory domain and the C-terminal tetra-
merization domain, was as expected [30] found to be more
resistant to proteolysis. When this enzyme form was
incubated with either H
4
biopterin, L-Phe, or both ligands
combined, no significant effect of the ligands on the rate of

proteolysis was observed as compared to the ligand-free
enzyme (Fig. 1B). In this case, the results from SDS/PAGE
analyses were confirmed by reversed-phase chromatogra-
phy of the peptides released during incubation (data not
shown).
Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 983
Effect of H
4
biopterin/H
2
biopterin on the conformational
transition (hysteresis) triggered by substrate binding
as studied by surface plasmon resonance analysis
The effect of the pterin cofactors on the reversible global
conformational transition induced by L-Phe binding to
tetrameric wt-hPAH was measured in real time by the time-
dependent change in refractive index (i.e. as a surface
plasmon resonance (SPR) response) of the immobilized
enzyme [25–27]. Approximately 25 ngÆmm
)2
(0.48 pmol
subunitÆmm
)2
) of tetrameric wt-hPAH was immobilized to
the dextran matrix of the sample surface, and a slightly
lower amount of the dimeric catalytic core enzyme
hPAH(Gly103–Gln428) (0.38 pmol subunitÆmm
)2
)was
immobilized to the reference surface [27]. The time-

dependent conformational change (SPR response) of the
full-length wt-hPAH was measured as a function of L-Phe
concentration and a steady-state (3 min response) binding
isotherm was obtained [26,27]. The isotherm observed in the
absence of biopterin cofactor was hyperbolic with a
concentration of L-Phe at half-maximum saturation
([S]
0.5
)of98±7l
M
(Fig. 2A,B). Saturation was reached
at approximately 2 m
M
with a DRU-value of 75 RU/(pmol
subunitÆmm
)2
). Simultaneous injection of L-Phe (variable
concentration) and 100 l
M
of H
4
biopterin resulted in a
 50% decrease in the maximum SPR response to L-Phe
(Fig. 2A), and the [S]
0.5
-value for L-Phe increased to
178 ± 11 l
M
. The presence of 500 l
M

of the oxidized
cofactor H
2
biopterin in the running buffer also lowers the
Fig. 2. The effect of pterin cofactor on the global conformational
transition of tetrameric wt-hPAH triggered by L-Phe binding as studied
by surface plasmon resonance. The effect of increasing L-Phe concen-
tration in the absence (d) and presence (s) of the pterin cofactor.
(A) 100 l
M
H
4
biopterin was coinjected with L-Phe. (B) 500 l
M
of the
soluble H
2
biopterin was included in the running buffer. The response
in the absence of pterin cofactor represents the average of two separate
titration experiments with a basal mean DRU-value of 25090 RU
corresponding to 0.12 pmol (0.48 pmol subunit) of immobilized
enzyme. The truncated form hPAH(Gly103-Gln428) was present on
the reference surface [27].
Fig. 1. The effect of ligand binding on the limited proteolysis by trypsin
of full-length wt-hPAH and the truncated form hPAH(Gly103-Gln428).
(A) Tetrameric wt-hPAH (24 induction with IPTG) preincubated with
either no ligand (d), 40 l
M
H
4

biopterin (m), 40 l
M
dopamine (n),
40 l
M
H
4
biopterin and 1 m
M
L-Phe (j), 40 l
M
dopamine and 1 m
M
L-Phe (h)or1m
M
L-Phe (s) before being subjected to limited pro-
teolysis by trypsin. (B) hPAH(Gly103-Gln428) was preincubated with
either no ligand (d), 40 l
M
H
4
biopterin and 1 m
M
L-Phe (j)or1m
M
L-Phe (s) before being subjected to limited proteolysis by trypsin. The
ratio hPAH : trypsin was 200 : 1 (m/m). The reactions were allowed to
proceed for up to 1 h at 25 °C and aliquots were taken at different time
intervals. The reaction was stopped by the addition of soybean trypsin
inhibitor (the ratio trypsin : inhibitor 1 : 1.5, m/m) and finally sub-

jected to SDS/PAGE stained with Coomassie Brilliant Blue. The gels
were scanned using
DESKSCAN II
(Hewlett Packard Co.); the volume of
the bands was analyzed by using the
PHORETIX
1
D
analysis software
from Nonlinear Dynamics Ltd, 1996 [10].
984 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003
maximum SPR response to L-Phe, most significantly
observed at concentrations above 200 l
M
(Fig. 2B), and
in this case, the [S]
0.5
-value for L-Phe increased to
123 ± 6 l
M
. It should be noted that in separate binding
experiments with H
4
biopterin alone the [S]
0.5
-value for the
cofactor was measured to 5.6 ± 0.8 l
M
with a D R-value
of 25 RU/(pmol subunitÆmm

)2
) at saturation [26].
Effect of active site ligands on the rate
of phosphorylation of recombinant hPAH
H
4
biopterin and L-Phe have been shown to inhibit and
stimulate, respectively, the rate of in vitro phosphorylation
of Ser16 by PKA in rPAH at physiologically relevant
concentrations [12]. In the present study, the ligand effects
on the phosphorylation of Ser16 by PKA in tetrameric
wt-hPAH were measured for the reduced cofactor H
4
biop-
terin, the oxidized cofactor H
2
biopterin and the catechol-
amine inhibitor dopamine. From Fig. 3A it is seen that on
preincubation with saturating concentrations (Fig. 3B) of
the biopterin cofactors or dopamine the rate of phosphory-
lation was decreased, and at 200 l
M
of the ligands the
inhibition was more pronounced for H
4
biopterin ( 26%)
and dopamine ( 26%) than for H
2
biopterin ( 12%). The
presence of L-Phe during preincubation with H

4
biopterin or
dopamine completely reversed their inhibitory effects on
phosphorylation (Fig. 3A). From Fig. 3B it is also seen that
the most potent inhibitor was dopamine, with a half-
maximum inhibition at < 0.1 l
M
,andH
4
biopterin ([I ]
0.5
of
1.4 l
M
, r ¼ 0.96) was more efficient than H
2
biopterin ([I ]
0.5
of 15.8 l
M
, r ¼ 0.86). The oxidized cofactor reached only a
 12% inhibition of phosphorylation compared to the
 26% for H
4
biopterin and dopamine. Interestingly, the
inhibitory effect of both biopterin cofactors revealed an
apparent negative cooperativity, with a Hill coefficient (h)of
0.46 (r ¼ 0.96) for H
4
biopterin (Fig. 3B, insert) and

h ¼ 0.75 (r ¼ 0.86) for H
2
biopterin. Hill coefficients < 1
were also observed for the inhibition of the phosphorylation
of the tetrameric fusion protein MBP-(pep)-hPAH, and
enzyme preparations isolated after a short (2 h) induction
period gave reproducibly a higher Hill coefficient with
H
4
biopterin than those isolated after a long (24 h) induction
period (data not shown). The relative efficiency of the
inhibition by the two biopterin cofactors is in good
agreement with the previously reported apparent K
d
values
for H
4
biopterin (0.09 ± 0.01 l
M
)andH
2
biopterin
(1.1 ± 0.02 l
M
) in their binding to rPAH, as determined
by fluorescence quenching titration [14]. The reduction of
the active-site iron [Fe(III) fi Fe(II)] by H
4
6-methyl-pterin
[14] prior to the phosphorylation assay, did not demonstrate

any significant effect on the apparent binding parameters
for H
4
biopterin in the present study. Moreover, we have
confirmed our previous finding with rPAH as the substrate
[12] that the dihydroxypropyl side-chain is required for the
cofactor inhibition of phosphorylation (data not shown).
The molecular basis for the negative modulatory
effects of the pterin cofactor and dopamine binding
to wt-hPAH and their reversal by substrate
The recently solved high resolution crystal structures of
hPAH and rPAH [16–21] have provided a detailed picture of
the protein contacts involved in the active site binding of the
pterin cofactor [18,20,21], dopamine inhibitor [17] and
L-Phe [21]. Thus, the superposition of the hPAH catalytic
core structures of the binary complexes with oxidized [18] or
reduced [20] pterin cofactor onto the structure of the ligand-
free rPAH (containing the regulatory and catalytic domains)
[19] revealed that both the reduced and the oxidized cofactor
interact with the N-terminal autoregulatory sequence at
Ser23. A close-up of this site of interaction (Fig. 4A,B) shows
that the O1¢ and O2¢ of the dihydroxypropyl side-chain
Fig. 3. The effect of H
4
biopterin, H
2
biopterin, dopamine and L-Phe on
the phosphorylation of tetrameric wt-hPAH. (A) Time-course for the
phosphorylation of wt-hPAH (4 l
M

) at standard incubation condi-
tions at 30 °C, including 60 l
M
[c-
32
P]ATP and 25 n
M
C-subunit of
protein kinase A (PKA). At timed intervals, aliquots of the reaction
mixture were spotted on phosphocellulose strips [24] to measure the
amount of
32
P transferred to the substrate by scintillation counting.
The incubations contained no ligand (d), 40 l
M
H
4
biopterin (m),
40 l
M
dopamine (n)or1 m
M
L-Phe in combination with either 40 l
M
H
4
biopterin or 40 l
M
dopamine (h). (B) The effect of increasing
concentrations of H

2
biopterin (s), H
4
biopterin (m) and dopamine (n)
on the rate of phosphorylation (t ¼ 10 min). Each point in the curves
represents the average of four measurements. Insert: a conventional
Hill plot on the H
4
biopterin data is shown in the main figure.
Y ¼ (v
o
) v
x
)/(v
o
) v
min
), which is the fractional decrease of phos-
phorylation rate seen at the concentration x of H
4
biopterin. v
o
is the
rate in the absence of H
4
biopterin and v
min
is the rate at very high
concentrations of H
4

biopterin. The observed Hill coefficient (h)was
found to be 0.46 (r ¼ 0.96).
Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 985
are sufficiently close to form favourable hydrogen bonds to
the carbonyl oxygen of Ser23 (Table 1). However, it is
important to note that the dihydroxypropyl side-chain is
positioned slightly different in the two redox states of the
cofactor and that the distances from Ser23O to O1¢ and O2¢
are slightly different (Table 1) because of the different
positions and hybridizations of the C6 atom of the pyrazine
ring in the two redox states [18,20]. This diversity of the side-
chain position is likely to entail different hydrogen-bonding
patterns to Ser23 in the full-length enzyme explaining the
redox state dependent regulatory properties of the pterin
cofactor. Moreover, the superposition of the ternary struc-
ture [21] revealed a similar orientation of the pterin cofactor
as in the binary structures [18,20]. However, the reduced
cofactor was found to be displaced by 2.6 A
˚
(mean distance)
in the direction of the iron and Glu286 upon substrate
binding, and in addition, the hydrogen-bonding network for
the cofactor was slightly different when compared to the
binary structures [18,20]. This displacement of the cofactor
results in a loss of stabilizing hydrogen bonds between O1¢
and O2¢ of the dihydroxypropyl side-chain and Ser23O in
the autoregulatory sequence (Fig. 4C and Table 1) and thus
explaining the complete reversal of the pterin cofactor as a
negative effector (Figs 1 and 3A,B).
When the crystal structure of the hPAHÆadrenaline/

dopamine binary complex [17] was superimposed onto that
of the ligand-free rPAH containing the regulatory and
catalytic domains [19] the catecholamine main-chain is also
Fig. 4. Stereo view of the site of interaction
between the pterin cofactors and dopamine with
the regulatory domain. The figure was pro-
duced by superimposing the crystal structure
of ligand-free dimeric rPAH (PDB id code
1PHZ), which contains both the regulatory
and the catalytic domains (residues 1–429)
onto the catalytic core crystal structures of (A)
binary hPAH with bound H
2
biopterin (PDB
id code 1LRM) (B) binary hPAH with bound
H
4
biopterin (PDB id code 1J8U) (C) ternary
hPAH with bound H
4
biopterin and substrate
(PDB id code 1KWO) and (D) binary hPAH
with bound dopamine (PDB id code 5PAH).
The backbone of rPAH regulatory domain
and hPAH catalytic domain are shown in red
and green, respectively, while residues Ser23
and Ile25 are shown by ball-and-stick repre-
sentation. The figure was prepared using
MOLSCRIPT
[42].

986 T. Solstad et al.(Eur. J. Biochem. 270) Ó FEBS 2003
seen to interact with the N-terminal autoregulatory
sequence (Fig. 4D and Table 1). The dopamine nitrogen
interacts with the carbonyl oxygen of Ser23 and the
dopamine Ca atom interacts with the side-chain of Ile25.
Discussion
The catalytic activity of PAH is regulated by four main
mechanisms, i.e. by substrate (L-Phe) activation, biopterin
cofactor (H
4
biopterin/H
2
biopterin) inhibition, increased
catalytic efficiency on phosphorylation of Ser16 (for review,
see [31]) and activation by spontaneous nonenzymatic
deamidation of specific labile Asn residues [10]. Internal
protein dynamics are intimately connected with these
regulatory properties. Based on our previous steady-state
enzyme kinetic and phosphorylation studies on rPAH a
working model was proposed to explain three of these
regulatory properties [12], involving four main conforma-
tional states (isomers) of the enzyme. The four conforma-
tional states include a ground state for the ligand-free
enzyme, an activated state with bound substrate (L-Phe), an
inhibited state with bound H
4
biopterin and finally the state
of catalytic turnover, i.e. the ternary enzyme-substrate
complex. Recent crystal structure analyses of the catalyti-
cally active core enzyme in different ligand-bound forms

[16–21] and complementary biophysical studies (reviewed in
[31]) strongly support such a model. Thus, both H
4
biopterin
and L-Phe bind reversibly at the active site of the core
enzyme by an induced fit mechanism with defined protein
contacts and conformational states [21]. Moreover, PAH is
inhibited by the covalent binding of catecholamines, i.e. by
bidentate coordination to the active site iron [17]. Whereas
the inhibition of the catalytic activity by catecholamines is
well understood at the structural level [17], the molecular
mechanism of the inhibitory properties of the pterin
cofactor (unrelated to its effect as electron donor in the
hydroxylation reaction) has been a controversial issue and is
further discussed below.
On the pterin cofactor binding site
Shiman and coworkers [14] have suggested the presence of
several putative binding sites for the natural cofactor
H
4
biopterin. The proposed binding sites are a regulatory
site (outside the active site) that is responsible for the
observed inhibitory effects of H
4
biopterin binding and a
redox site responsible for the reduction of the active site iron
in addition to its binding at the catalytic site as part of a
catalytically active ternary complex. Based on the crystal
structure of a ligand-free dimeric C-terminal truncated form
of rPAH [19] and analogies with the structure of pterin-4a-

carbinolamine dehydratase (PCD/DCoH) a putative bind-
ing site for H
4
biopterin in the regulatory domain, close to a
proposed hinge region (residues 111–117), has been pro-
posed [19]. This region is also considered as the target for
L-Phe induced proteolytic cleavage by trypsin [30]. More-
over, the binding of L-Phe to a second putative site in the
regulatory domain was suggested to induce a conforma-
tional transition that modifies the intra-subunit interaction
between the regulatory and the catalytic domains. This
interaction is followed by the formation of a catalytically
activated form of the enzyme and an increased susceptibility
to tryptic cleavage. Thus, binding of H
4
biopterin to the
proposed regulatory site was suggested to prevent the
interdomain hinge-bending motion required for activation
and susceptibility towards proteolysis [19]. However, recent
crystal structure analyses of the binary complex with
H
2
biopterin [18] and the binary and ternary complexes
with H
4
biopterin [20,21] have defined the protein contacts
involved in cofactor binding at the active site and their
interactions through the dihydroxypropyl side-chain with
the autoregulatory sequence in the regulatory domain
(Fig. 4A,B). Thus, no direct structural evidence (by X-ray

or NMR) has so far been presented in support of a
H
4
biopterin (and L-Phe) binding site in the regulatory
domain [18–21,31,32]. Based on this structural information,
we here propose an alternative molecular mechanism for the
inhibitory effects of the biopterin cofactor on the rate of
phosphorylation of Ser16, the limited proteolysis by trypsin
and the substrate induced conformational transition (hys-
teresis) related to catalytic activation.
The inhibited forms of the enzyme with bound
biopterin cofactor or dopamine
The phosphorylation site in PAH (Ser16) is localized in the
N-terminal tail (residues 1–18) of the autoregulatory
sequence for which no interpretable electron density has
been obtained [19], compatible with a rather flexible
structure of the N-terminus [33]. Our experimental data
and the crystal structure analysis discussed above, show the
interactions of H
4
biopterin, H
2
biopterin and dopamine with
the N-terminal autoregulatory sequence at Ser23 and Ile25
(Fig. 4A,B,D; Table 1). We can now present an explanation
for the inhibitory effect of the biopterin cofactor and
dopamine on the rate of phosphorylation (Fig. 3), on limited
proteolysis (Fig. 1A) and on the global conforma-
tional transition (hysteresis) related to catalytic activation
Table 1. Comparison of distances (A

˚
) of the superposition of the crystal structure of ligand-free dimeric rPAH (PDB id code 1PHZ), which contains
both the regulatory and the catalytic domains (residues 1–429) onto the catalytic core crystal structures of binary hPAH with bound H
4
biopterin (PDB
id code 1J8U), binary hPAH with bound H
2
biopterin (PDB id code 1LRM), ternary hPAH with bound H
4
biopterin and substrate (PDB id code 1KW0)
and binary hPAH with bound dopamine (PDB id code 5PAH).
Distance from Ser23O to H
4
biopterin structure H
2
biopterin structure THA-H
4
biopterin structure Dopamine structure
H
4
biopterin O2¢ 2.4 3.8
H
4
biopterin O1¢ 3.0 5.4
H
2
biopterin O2¢ 4.0
H
2
biopterin O1¢ 1.3

Dopamine N1 3.0
Ó FEBS 2003 Regulatory properties of phenylalanine hydroxylase (Eur. J. Biochem. 270) 987
(Fig. 2A,B), i.e. by their direct binding at the active site. That
H
4
biopterin is a more potent inhibitor than H
2
biopterin of
the global conformational transition triggered by L-Phe
binding was most directly demonstrated by the SPR
analyses. Whereas 100 l
M
H
4
biopterin resulted in a
 50% reduction in the maximum SPR response to L-Phe
binding (Fig. 2A), H
2
biopterin gave only a minor reduction
(maximum  11%) at concentrations higher than 200 l
M
(Fig. 2B). Interestingly, the inhibitory effect of the biopterin
cofactor on the rate of phosphorylation revealed an
apparent negative cooperativity, with a Hill coefficient (h)
of  0.5 for H
4
biopterin (Fig. 3B, insert) and  0.8 for
H
2
biopterin. A negative cooperativity has also been reported

for the structurally and functionally related human enzyme
tyrosine hydroxylase in a direct binding assay [25]. Further-
more, the Hill coefficient for H
4
biopterin binding to hPAH
revealed a dependence (both for the isolated tetrameric
wt-hPAH and the tetrameric fusion protein MBP-(D
4
K)
ek
-
hPAH) on the induction time with IPTG in E. coli,i.e.a
short induction period (2 h at 28 °C) gave a slightly higher
Hill coefficient than 24 h induction (data not shown). This
finding may be related to the differences observed in our
steady-state kinetic analyses of the two enzyme forms [10].
Thus, the tetrameric wt-hPAH isolated after 2 h and 24 h of
induction with IPTG in E. coli, revealed differences (24 h vs.
2 h) in both the affinity for the cofactor H
4
biopterin
(decreased) and L-Phe (increased), as well as for the catalytic
efficiency (increased) [10]. The main physico-chemical
difference between these two enzyme preparations was the
extent of time-dependent nonenzymatic deamidation (dur-
ing expression in E. coli) of specific labile Asn residues [10].
Complementary mutagenesis (AsnfiAsp) analyses have
demonstrated that the rate of phosphorylation is indeed
dependent on the extent of deamidation of a very labile Asn
residue (Asn32) in the N-terminal autoregulatory sequence

of wt-hPAH [34].
Dopamine has a dual mechanism for its inhibition of
PAH. First, the bidentate coordination of its catechol
hydroxyl groups to the active site iron [17] results in a
complex with strong inhibition of the catalytic activity [32].
Secondly, on binding at the active site, the dopamine main-
chain interacts with the autoregulatory sequence at Ser23
(by hydrogen bonding) and Ile25 (Fig. 4), which results in
an inhibition of Ser16 phosphorylation similar to that of
H
4
biopterin (Fig. 3B) with a half-maximum inhibition at a
concentration < 0.1 l
M
. This value compares well with the
concentration determined for the half-maximum binding
(0.25 l
M
) of noradrenaline to rPAH [32]. A similar type of
interaction between dopamine and the regulatory domain of
the structurally and functionally closely related enzyme
tyrosine hydroxylase has been reported in experiments using
limited proteolysis as a structural probe [35]. In this case, the
affinity of dopamine binding was even higher than in PAH,
with a K
d
¼ 1.3 ± 0.6 n
M
, which is considered to be of
physiological relevance (reviewed in [36]).

The interdependent binding of biopterin cofactor
and amino acid substrate
Internal protein dynamics are intimately connected to the
catalytic activity of PAH, and our recent structural studies
on hPAH have revealed some key features of its hysteretic
properties. Thus, the structures of the binary and ternary
complexes of hPAH have provided a detailed picture of the
protein contacts involved in the binding of biopterin
cofactor and amino acid substrate at distinctly different
positions in the active site crevice structure [20,21]. More-
over, the cystal structures have revealed that the binding of
both biopterin cofactor and substrate is accompanied by
local (active site) conformational changes, most pronounced
for the binding of L-Phe, which also triggers global
conformational changes (hysteresis) in the protomer as
determined by SPR analyses (Fig. 2 [26,27]). However, as
there is still no crystal structure available for the full-length
form of the ligand-free enzyme and the binary substrate
complex, it is not known how the observed conformational
changes at the active site [21] are propagated to the rest of the
full-length protomer in the oligomeric forms. The structural
changes observed at the active site of the catalytic domain
structure [21] explain why the binding of L-Phe reduces the
affinity of pterin cofactor binding (Fig. 4C) and vice versa
(Fig. 2). Thus, the large conformational change observed at
the active site on substrate binding changes the position and
orientation (relative to the catalytic iron) and the hydrogen-
bonding network of the bound H
4
biopterin. Moreover, the

substrate induced repositioning of the cofactor also accounts
for the release of its interaction with the autoregulatory
sequence (Fig. 4C) and the related inhibition of Ser16
phosphorylation (Fig. 3). A similar effect of substrate
binding was observed for the dopamine inhibition of
phosphorylation (Fig. 3) and of the limited proteolysis by
trypsin (Fig. 1), which are both completely reversed by
L-Phe binding. The interdependent binding properties of the
pterin cofactor and L-Phe have also been observed in kinetic
studies on mutant forms of hPAH (point mutations of active
site residues), which often have a profound influence on
biopterin cofactor and/or substrate binding affinity [18,
37–40]. If the affinity for the cofactor is reduced, that of the
substrate is often observed to be increased, and vice versa
[18,40]. It should be noted that enzyme kinetic, three-
dimensional structural and biophysical (MCD and EXAFS)
studies all support an ordered reaction mechanism wherein
both cofactor and substrate must be bound before reaction
with dioxygen can occur to generate the active intermediate
for the coupled hydroxylation of the cosubstrates [21,41].
However, the sequence of binding of cofactor and substrate
is still a matter of discussion and may be different comparing
wild-type and mutant forms of the enzyme.
Acknowledgements
The study was supported by grants from the Research Council of
Norway (NFR), from The Novo Nordisk Foundation, The Nansen
Fund, The Blix Family Fund for Advancement of Medical Research
and the Norwegian Council on Cardiovascular Diseases. We greatly
appreciate the expert technical assistance of Ali Sepulveda Mun
˜

oz in
expression and purification of the recombinant enzymes, and the staff
of the Swiss-Norwegian Beamlines in Grenoble (France).
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