Despite the similarities between THand PAHregarding the structure of the active site and the catalytic mecha-nism, there is one striking difference: THaccepts also phenylalanine as subst
Trang 1Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms
of phenylalanine hydroxylase and tyrosine hydroxylase
Astrid Maaß1, Joachim Scholz2,3and Andreas Moser2
1
Fraunhofer-Institute for Algorithms and Scientific Computing (SCAI), Schloss Birlinghoven, Sankt Augustin, Germany;
2
NeurochemistryResearch Group, Department of Neurology, Medical Universityof Lu¨beck, Lu¨beck, Germany;
3 Neural PlasticityResearch Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital
and Harvard Medical School, Charlestown, Massachusetts, USA
NMR spectroscopy and X-ray crystallography have
provi-ded important insight into structural features of
phenyl-alanine hydroxylase (PAH) and tyrosine hydroxylase (TH)
Nevertheless, significant problems such as the substrate
specificity of PAHand the different susceptibility of TH
to feedback inhibition by L-3,4-dihydroxyphenylalanine
(L-DOPA) compared with dopamine (DA) remain
unre-solved Based on the crystal structures 5pah for PAHand
2toh for TH(Protein Data Bank), we have used molecular
docking to model the binding of 6(R)-L
-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the substrates phenylalanine
and tyrosine to the catalytic domains of PAHand TH
The amino acid substrates were placed in positions common
to both enzymes The productive position of tyrosine
in THÆBH4was stabilized by a hydrogen bond with BH4
Despite favorable energy scores, tyrosine in a position trans
to PAHresidue His290 or THresidue His336 interferes with the access of the essential cofactor dioxygen to the catalytic center, thereby blocking the enzymatic reaction DA and
L-DOPA were directly coordinated to the active site iron via the hydroxyl residues of their catechol groups Two alter-native conformations, rotated 180 around an imaginary iron–catecholamine axis, were found for DA andL-DOPA
in PAHand for DA in TH Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction productL-DOPA to TH, thereby saving the enzyme from direct feedback inhibition
Keywords: phenylalanine hydroxylase; tyrosine hydroxylase; substrate specificity; catecholamines; feedback inhibition
Phenylalanine hydroxylase (PAH, EC 1.14.16.1), tyrosine
hydroxylase (TH, EC 1.14.16.2), and tryptophan
hydroxy-lase (TPH, EC 1.14.16.4) constitute a family of closely
related aromatic amino acid hydroxylases sharing structural
as well as functional features [1,2] The three enzymes are
each composed of an N-terminal regulatory domain and a
C-terminal region containing a highly conserved catalytic
core domain and a tetramerization domain [3] Studies using
partial proteolysis or heterologous expression of truncated
enzymes have shown that the C-terminal amino acids 165–
479 of rat TH[4] and the C-terminal residues 142–410 of rat PAH[5] retain the catalytic activity of these enzymes Sequence comparison reveals that the catalytic domains of THand PAHpossess 65% sequence identity and 80% homology [3] (Fig 1) In a catalytic mechanism shared by PAHand TH, an aromatic amino acid is hydroxylated within the highly conserved active site containing a single, iron(II) atom Dioxygen and 6(R)-L -erythro-5,6,7,8-tetra-hydrobiopterin (BH4) are essential cosubstrates of the reaction A coupled hydroxylation of the amino acid and the pterin takes place after all three substrates (BH4, dioxygen, amino acid) have bound to the active site [6,7] THand PAHare subject to feedback inhibition by
L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine (DA), noradrenaline and adrenaline These end products of catecholamine synthesis are competitive inhibitors vs BH4 and lead to oxidation of the catalytic iron [8,9]
Analyses of truncated forms of rat TH, rat and human PAHby means of X-ray crystallography have provided insight into the three-dimensional structure of the catalytic domains of the two enzymes [10–13] X-ray crystallography
of 7,8-dihydrobiopterin (7,8-BH2) bound to truncated rat THand human PAHhas identified amino acid residues critical for the positioning of this oxidized cosubstrate in the second coordination sphere of the catalytic iron [13,14] The impact of the structural identity of the pterin cosubstrate on THactivity has been shown in a kinetic study using synthetic pterin analogs [15] Spectroscopic investigations of
Correspondence to A Maaß, Fraunhofer Institute for Algorithms
and Scientific Computing (SCAI), Schloss Birlinghoven,
53754 Sankt Augustin, Germany.
Fax: + 49 2241 142656, Tel.: + 49 2241 142481,
E-mail: astrid.maass@scai.fhg.de
Abbreviations: BH 4 , 6(R)- L -erythro-5,6,7,8,-tetrahydrobiopterin;
7,8-BH 2 , 7,8-dihydrobiopterin; DA, dopamine; L -DOPA,
L -3,4-dihydroxyphenylalanine; PAH, phenylalanine hydroxylase;
TH, tyrosine hydroxylase.
Enzymes: aromatic L -amino acid decarboxylase (EC 4.1.1.28);
phenylalanine hydroxylase (phenylalanine-4-hydroxylase;
EC 1.14.16.1); tryptophan hydroxylase (tryptophan-5-monooxygenase;
EC 1.14.16.4); tyrosine hydroxylase (tyrosine-3-monooxygenase;
EC 1.14.16.2).
(Received 9 October 2002, revised 29 November 2002,
accepted 16 December 2002)
Trang 2THand crystallographic data obtained from binary
com-plexes of catecholamines and truncated human PAHhave
demonstrated that the two hydroxyl groups of the catechol
moiety bind to the catalytic iron [16–18]
Despite the similarities between THand PAHregarding
the structure of the active site and the catalytic
mecha-nism, there is one striking difference: THaccepts also
phenylalanine as substrate, with Kmincreased by a factor
of six and Vmaxdecreased by a factor of four compared
with tyrosine [19] In contrast, PAHis not able to further
hydroxylate its product tyrosine Mutation studies have
revealed the significance of single amino acids or larger
portions within the catalytic domain for substrate affinity
and substrate specificity of PAHand TH[19–21]
However, it still remains unresolved as to which actual
features of the structural environment defining the active
site underlie the substrate specificity of PAHand TH
Rapid conversion of active cosubstrates and substrates
into their products makes it difficult to produce complexes
that are sufficiently stable to be crystallized or undergo
NMR spectroscopy Recently, molecular modeling based
on crystal structures [22] or NMR spectroscopy [23] of
PAHwith bound substrate analogs has been employed to
elucidate ligand binding to the active site of this enzyme
Multiple sequence alignment and knowledge of the crystal
coordinates of PAHand THhas been used to model the
full length structure of TPH[24]
We have modeled the catalytic sites of PAHand THand
introduced BH4 and the amino acid substrates
phenyl-alanine or tyrosine by molecular docking in order to
investigate structural properties responsible for the
differ-ence in the substrate specificity of the enzymes In a separate
set of docking experiments, we modeled the inhibition of
PAHand THby catecholamine end products to explain the
reduced susceptibility of THto feedback inhibition by
L-DOPA compared with DA
Experimental procedures
Ligand–protein complexes were generated based on the
crystal structures 5pah for PAHand 2toh for TH(Protein
Data Bank) [13,17] The softwareFLEXX, version 1.7.6 was
used for ligand docking [25] The complexes were optimized
by force-field energy minimization using , version
23.2 [26].CHARMMandCAMLAB, version 1.0 were applied to calculate the total energy in aqueous solution [27] Construction of ligand–protein complexes The active sites in the docking runs included all atoms within a radius of 8.0 A˚ around the reference ligands 7,8-BH2 or DA in the crystal structures 2toh or 5pah, respectively Iron(II) was parametrized for FLEXX as a divalent cation Assuming that dioxygen replaced one of the iron-bound crystal water molecules [13,17], one of these water molecules served as a placeholder within the iron coordination sphere THresidue 300, specified as meta-tyrosine, was reverted to phenylalanine, as this residue has been hydroxylated artificially during crystallization [28] The positions of crystal water molecules within the coordi-nation sphere and of hydrogen atoms added to the protein structure were optimized by 100 steps of conjugate gradient energy minimization with the dielectric constant e¼ 2r and convergence ensured throughout
Ligand structures were divided into fragments and reconstructed stepwise within the active site usingFLEXX
As alternative placements of the ligand fragments are possible, a set of conformations resulted, which were ranked based on their energy score [29] Placements close to the true conformation are supposed to have low energy and will occupy the top ranks
Energy minimization Each complex was subjected to 600 steps of conjugate gradient energy minimization (e¼ 2r) Ligands and iron-bound water molecules were allowed to move freely, whereas the protein and the iron atom were fixed Atomic partial charges of the ligands were calculated using the Charge-Templates method (Quanta, MSI) A cut-off value
of 15 A˚ was applied in the computation of coulombic interactions
Energy calculation The energy-minimized structures were re-ranked according
to their total energy in aqueous solution The total energy was modeled as the sum of theCHARMMforce field energy in
a homogeneous dielectric medium (e¼ 4) plus the solvation energy calculated byCAMLAB The nonpolar contribution to the solvation energy was assumed to be proportional to the solvent-accessible surface of the complex with a surface tension constant of 84 JÆA˚)2 This value is derived from the distribution coefficients for alkanes in polar and nonpolar solvents [27] The polar portion was estimated by solving the Poisson–Boltzmann equation [30,31] twice using a fast multigrid finite difference solver [32] First, the electrostatic energy was calculated for a heterogeneous system with the dielectric constants einternal¼ 4 inside the molecular surface
of the complex, and eexternal¼ eH2O¼ 78.5 outside The molecular surface was defined by the van der Waals radii of atoms composing the complex Secondly, the electrostatic energy was computed assuming a homogeneous dielectric system, with the dielectric constants einternal¼ eexternal¼ 4 The electrostatic contribution to the solvation energy was obtained by the difference between the two electrostatic
Fig 1 Alignment of amino acid residues composing the catalytic
domains of PAH and TH Residues identical in both enzymes are
highlighted Dark gray bars indicate amino acid residues that possess
at least one atom within a distance of 14 A˚ from the catalytic iron and,
based on this criterion, were included in energy calculations.
Trang 3energies The total electric charge was +6 for 5pah and)16
for 2toh, as 2toh comprises the catalytic core domain and
the tetramerization domain BH4is uncharged The
aroma-tic amino acids phenylalanine and tyrosine were
implemen-ted in their zwitterionic state with a protonaimplemen-ted amine group
and a carboxylate moiety, resulting in a total charge of zero
DA possesses an electric charge of +1
We restricted the region of the complex for which the
total energy was calculated to those amino acid residues and
molecules that had at least one atom within a radius of 14 A˚
around the iron atom This sphere included about 50% of
the catalytic domain and comprised the entire binding
pocket containing the ligand (Fig 1)
Analysis of results
For each ligand-protein pair, this procedure led to a set of
200–300 diverse structure predictions and relative total
energies Considering that the relevant parts of the
con-formational space were probed and that the relative total
energy is a reasonable approximation of the free energy, the
structure with the lowest total energy should be closest to
the true structure of the complex in solution However,
conformations with low energy values were discarded if the
predicted ligand position extended into the regulatory
domain of PAH[33] Assuming that all reactive ligand
groups are placed in close proximity to the active site iron
immediately before the enzymatic reaction, only placements
within a defined ligand-iron distance were considered
relevant According to the X-ray crystallographic structures
of THand PAHwith bound 7,8-BH2 (2toh, 1dmw), the
carbonyl oxygen of 7,8-BH2is the atom closest to the active
site iron with a distance of 3.6 or 3.8 A˚, respectively
Therefore, conformations of BH4with a maximal distance
dBH4-Feof 4.5 A˚ were included in further analyses (Fig 2)
As an oxygen atom may be placed between the ring of the
amino acid substrates and the active site iron, the maximal
distance dTyr-Feor dPhe-Febetween the center of the ring and
the iron atom was defined as 6.5 A˚ (Fig 2) As previous
spectroscopic and X-ray crystallographic studies have suggested a tight bidentate coordination of catecholamines towards the iron atom, 5.0 A˚ was set as the maximal distance dL -DOPA-Fe or dDA-Fe between an imaginary line connecting the oxygen atoms of the two catechol hydroxyl groups and the iron (Fig 2) This distance criterion would allow bidentate, monodentate and other binding modes to
be included in further analysis
Results
Pterin binding to the catalytic domains of PAH and TH Docking of the native cosubstrate BH4 into the crystal structure of the PAHcatalytic domain yielded a total of
286 conformations Out of these, 44 conformations were considered relevant as the distance between the pterin carbonyl oxygen and the PAHcatalytic iron atom (dBH4-Fe) was less than 4.5 A˚ (Fig 3) The energetically most favorable conformer corresponded with the position of 7,8-BH2bound to PAHat the first coordination sphere of the iron atom as previously determined by NMR spec-troscopy (rmsd 2.4 A˚) [23] and X-ray crystallography (rmsd 2.1 A˚) [14] The pterin backbone was close to the aromatic ring of PAHresidue Phe254, with a relative tilt of about 20 The guadinium moiety was anchored by an H-bond between N1 and the amine group of Leu249 The distance between N4 and the side chain of Glu286 was 4.6 A˚ This allows a putative water molecule to be placed
in between, which stabilizes the complex by additional H-bonds (Fig 3)
Docking BH4into the crystal structure of THproduced
300 conformations; in 46 out of these, the distance dBH4-Fe was shorter than 4.5 A˚ (Fig 3) The energetically most favorable placement was very similar to the conformation of
BH4 in PAH(rmsd 2.3 A˚) The pterin ring was in close contact to Phe300 with the two ring planes tilted by 32 The carbonyl oxygen of BH4coordinated directly to the iron and the guanidinium moiety was fixed by an H-bond between the proton of N2 and the backbone oxygen of Gln310 (Fig 3) This conformation of BH4 coincided with the conformation of BH4in THpreviously computed by Alma˚s
et al usingDOCK4.0 (rmsd 2.2 ± 0.2 A˚) [15] However, it differed from the conformation of 7,8-BH2 in the binary complex identified by X-ray crystallography [13] In the latter study, the position of 7,8-BH2was rotated by 180 and characterized by a p-stacking interaction of the planar pterin moiety with THresidue Phe300, tilted by 10
We manually docked BH4into THto further investigate this alternative pterin position The energetically most favorable manual placement was in good agreement with the rotated position of 7,8-BH2cocrystallized in TH(rmsd 1.5 A˚) The pterin backbone was close to the aromatic ring
of Phe300, tilted by 45, and the carbonyl oxygen was again coordinated towards the iron atom The 2-hydroxyl group
of BH4 formed an H-bond to the carboxylate group of Glu332 The distance dBH
4 -Fewas 2.27 A˚, thus similar to 2.30 A˚ in the conformer obtained by automated docking (Fig 3) The lowest total energy in the group of manually docked conformations was 11.4 kcalÆmol)1compared with 1.5 kcalÆmol)1for the most favorable conformer in auto-mated docking (Fig 3) The difference mainly resulted from
Fig 2 Ligands docked into the crystal structure of PAH and TH.
Placements suggested by FLEXX were considered relevant and included
in further analyses if the indicated distances between the ligands and
the iron atom at the active site of the enzymes were within defined
limits.
Trang 4presumably artificial straining of the deeply buried BH4
-sidechain, caused by the minimization conditions applied
Hence both rotamers should be treated as equivalent The
equivalence of the two conformations was underscored by
the fact that the orientation of the pterin cosubstrate did not
affect the subsequent placement of amino acid substrates in
the ternary complexes with TH
The position of amino acid substrates in the complexes
with PAHÆBH4and THÆBH4
After docking the native substrate phenylalanine into
PAHÆBH4, 73 candidate positions with a distance dPhe-Fe
between the center of the aromatic ring and the THiron not
exceeding 6.5 A˚ were included in further analysis In the
conformation with the lowest total energy (13.5 kcalÆmol)1),
the distance between the phenylalanine ring center and the
iron atom was 4.96 A˚ The carboxylate moiety of
phenyl-alanine was anchored by H-bonds to PAH residue Arg270
Another H-bond was formed between the carboxylate
group of phenylalanine and the amine group of Thr278 The
ammonium group of phenylalanine formed an H-bond to
the carbonyl oxygen of Thr278 (Fig 4) Table 1
summar-izes relevant energy components for the interactions of
phenylalanine in the complex with PAHÆBH4 This
phenyl-alanine position provided by FLEXX and the calculated
hydrogen bonds to surrounding PAHresidues are in
agreement with X-ray crystallographic data of the phenyl-alanine analog 3-(2-thienyl)-L-alanine bound to PAH[22]
In another, energetically equivalent conformation the phenyl ring occupied the same position but the ammonium group now formed an H-bond with Ser349, while the salt bridge between the carboxylate group and Arg270 is maintained (not shown) This latter position matches the conformation of phenylalanine in the complex with PAHÆ7,8-BH2that was previously calculated after restraints from NMR spectroscopy [23] (rmsd 2.09 A˚ and 1.29 A˚, respectively)
Docking of tyrosine into PAHÆBH4produced a set of 179 conformations Out of these, 55 conformations fulfilled the distance criterion of dTyr-Febeing smaller than 6.5 A˚ Only eight conformations displayed the expected coordination of the tyrosine aromatic ring towards the iron atom However, the hydroxyl group of the aromatic ring was placed trans to His290 In contrast to the anchoring of the native substrate phenylalanine, the ammonium group of tyrosine formed an H-bond with the carboxyl moiety of Pro279 (Table 1) As shown in Fig 4, this position of tyrosine in PAHdiffered significantly from that of phenylalanine
FLEXXprovided 318 possible conformations of tyrosine in THÆBH4 A set of 150 conformations was regarded as relevant The position with the lowest total energy corres-ponded to the conformation of tyrosine in PAH(rmsd 1.12 A˚) In this position, the hydroxyl moiety of tyrosine
Fig 3 Placement of BH 4 in the catalytic site of PAH and TH Distance-energy diagrams obtained after docking BH 4 into the active sites of PAH(A) and TH(B) Total energies of candidate positions provided by automated FLEXX calculations are given as s, total energies of conformations obtained
by manual docking are shown as· (C) Superposition of BH 4 placements in the catalytic sites of PAH and TH Enzyme (PAH/TH) residues mentioned
in the text are displayed based on Protein Data Bank files 5pah and 2toh by using the program RASMOL (Sayle, R., Glaxo Wellcome Research and Development, Stevenage, Hertfordshire, UK) Protein structures are depicted as sticks with carbon atoms colored purple, nitrogen atoms blue and oxygen atoms red The iron at the center of the active side is colored orange Atoms coordinating directly to the iron atom are shown as balls The top-scoring conformation obtained by FLEXX for BH 4 in PAHis shown in red, for BH 4 in THaccording to the results of the automated docking in blue, and for BH 4 in THaccording to the manual docking in the X-ray crystallographic mode in green Hydrogens are omitted for clarity.
Trang 5was again oriented towards the catalytic iron atom of TH, with a distance of 2.16 A˚ between the hydroxyl oxygen and the iron In analogy to the position of tyrosine in PAHÆBH4, the hydroxyl group was placed trans to THresidue His336 The ammonium group of tyrosine formed an H-bond to Asp425 (Table 2) As pointed out below, this position is likely to hinder the access of the essential reaction cofactor dioxygen to the catalytic center In contrast to the placement
of tyrosine in PAHÆBH4however,FLEXXprovided a second conformation for tyrosine, which was almost identical to the position predicted for the native substrate phenylalanine in the complex with PAHÆBH4 This conformation was characterized by a salt bridge between the carboxylate group and the guanidinium moiety of Arg316 (Table 2) The ammonium group of tyrosine was surrounded by the backbone oxygens of THresidues Ser324 and Pro325 (Fig 4) The orientation of the pterin cosubstrate in the complex with THdid not have an effect on the position of tyrosine However, the orientation of BH4 in the second-ranking conformation of tyrosine in THÆBH4corresponded
to that of 7,8-BH2cocrystallized with TH[13] and in this orientation, N3 of BH4exhibited a stabilizing H-bond to the tyrosine hydroxyl group
The best placement of phenylalanine in THÆBH4 coincided with the second-ranking position of tyrosine, and it also corresponded to the position obtained by phenylalanine in PAHÆBH4(rmsd 1.65 A˚) Forty-eight out
of 319 calculated conformations were considered relevant here In the energetically most favorable position (total energy 1.3 kcalÆmol)1), the center of the aromatic ring was placed at a distance of 5.86 A˚ from the active-site iron, compared with 4.95 A˚ for tyrosine in THÆBH4 (Fig 4) The increase in the distance is caused by an additional iron-bound water molecule required for the docking of phenylalanine
Table 1 Intermolecular interaction energy contributions for the relevant amino acid placements in PAHÆBH 4 Energy values for van der Waals interactions (E vdW ), coulombic (electrostatic) interactions (E Coulomb ) and H-bonds of the ligand to neighboring protein residues are given in kcalÆmol)1.
PAHresidue
Phenylalanine in PAHÆBH 4 Tyrosine in PAHÆBH 4
Arg270 – 1.12 – 6.04 – 0.90 Leu248 – 0.75 – 0.21
Met276 – 0.74 1.16 Pro279 – 1.38 – 2.13 – 2.53 His277 – 1.45 – 1.10 Glu280 – 1.54 – 0.28
Thr278 – 2.65 – 4.41 – 3.00 Pro281 – 3.20 – 0.16
Glu280 – 1.31 – 1.36 Trp326 – 1.48 0.07
Pro281 – 1.73 – 0.09 Glu330 – 1.92 2.20
Asp282 – 0.37 0.57 Val379 – 1.28 – 0.39
His285 – 3.16 – 0.08
Glu330 – 1.08 – 1.80 Iron atom – 17.32 – 15.86
Phe331 – 0.85 0.09 BH 4 – 2.78 – 1.62 – 2.69 Gly346 – 1.50 0.09
Ser349 – 1.85 1.08
Ser350 – 1.41 0.10
Val379 – 0.12 – 0.38
Iron atom – 0.04 2.90
Fig 4 Amino acid substrates docked into complexes of PAH or TH
with bound BH 4 Superimposed are the top-scoring confomations of
phenylalanine in PAH(light green) and tyrosine in PAH(red) The
tyrosine conformation in THwith the lowest total energy is shown in
darker green; the second-ranking tyrosine conformation (blue)
cor-responds closely to the position of phenylalanine in PAH The
place-ment of phenylalanine in THis given in light green BH 4 in the
corresponding complexes is shown in the same color as the amino acid
substrate.
Trang 6Differences in the binding ofL-DOPA and DA
to the active site iron of PAH and TH
In agreement with previous results from X-ray
crystallo-graphy [17], a common mode of bidentate binding was
found when the catecholamine end productsL-DOPA and
DA were docked into PAH The two hydroxyl groups of
the catechol moiety formed a tight chelate complex with
the iron atom at the center of the active site.FLEXXyielded
189 conformations ofL-DOPA and 213 conformations of
DA in 5pah; 32 candidate positions forL-DOPA and 55
positions for DA were within a distance of 5.0 A˚ Nine
independent predictions forL-DOPA converged to the same
local minimum, which was characterized by an H-bond
between the amine group ofL-DOPA and the backbone
oxygen of PAHresidue Leu249, and a second H-bond
between the L-DOPA carboxyl moiety and the Leu249 nitrogen (Fig 5) The next favorable conformation was rotated by 180 around an imaginary axis passing between the two hydroxyl groups of the catechol moiety (Fig 5) The difference of 5.5 kcalÆmol)1in the total energy of the two orientations presumably represents an overestimate resulting from energy minimization in the absence of solvent molecules For DA, the second favorable conformation was also rotated by 180 Here, the difference between the two rotamers was 0.3 kcalÆmol)1, thus negligible Apparently, in PAHtwo equivalent positions exist forL-DOPA and DA, with the plane of the catechol ring rotated 180
The docking of DA into THyielded 216 relevant conformations DA bound directly to the active site iron (Fig 5) The amine end freely stuck out of the active site crevice, analogous to the placement of DA in PAH(rmsd
Table 2 Intermolecular interaction energies for the placement of tyrosine or phenylalanine in the complex of THÆBH 4 Energy contributions from van der Waals interactions (E vdW ), coulombic (electrostatic) interactions (E Coulomb ) and H-bonds between ligands and neighboring protein residues are shown Values are expressed in kcalÆmol)1.
Tyrosine in THÆBH 4
(unproductive position)
Tyrosine in THÆBH 4
(productive position)
Phenylalanine
in THÆBH 4
THresidue E vdW E Coulomb H-bonds TH residue E vdW E Coulomb H-bonds TH residue E vdW E Coulomb H-bonds Leu294 – 2.57 – 0.23 Arg316 – 1.06 – 4.78 Arg316 – 0.34 – 10.02 – 0.68 Pro325 – 1.52 – 0.16 Met322 – 0.36 0.68 Met322 – 0.14 0.22
Glu326 – 1.53 – 0.54 His323 – 2.19 – 1.85 His323 – 0.57 – 0.71
Pro327 – 2.91 – 0.03 Ser324 – 2.51 – 4.81 – 4.90 Ser324 – 1.57 – 3.88 – 3.31 His331 – 0.72 0.17 Pro325 – 1.88 – 1.44 Pro325 – 1.01 – 1.40
Trp372 – 1.53 0.04 Glu326 – 1.31 – 0.74 Glu326 – 1.52 – 0.08
Glu376 – 1.27 1.85 Pro327 – 1.85 – 0.24 Pro327 – 2.45 – 0.48
Asp425 – 0.40 – 13.12 – 1.03 Asp328 – 0.37 1.33 Asp328 – 0.49 3.68
His331 – 2.87 0.44 His331 – 2.97 – 0.75 Iron atom – 15.62 – 12.89 Glu376 – 1.62 0.39 Glu376 – 1.64 – 0.57
BH 4 – 2.95 1.00 Phe377 – 1.10 0.12 Phe377 – 1.11 0.10
Gly392 – 1.27 0.14 Gly392 – 1.16 0.16 Ser395 – 1.32 0.73 Ser395 – 2.38 1.00 Ser396 – 1.33 )1.04 Ser396 – 0.86 – 0.37 Asp425 – 0.19 )3.90 Asp425 – 0.33 – 3.17 Iron atom – 2.49 )4.26 Iron atom – 0.05 – 0.20
Fig 5 Binding of catecholamine end products at the catalytic site (A) High-scoring conformations of the catecholamines L -DOPA (blue, darker green) and DA (red, light green) in the catalytic site of PAH (B) High-scoring positions of L - DOPA (red) and DA (blue, green) docked into the catalytic site of TH.
Trang 72.15 A˚) Like in PAH, two conformations of DA in TH,
rotated 180 around their iron-catecholamine axis,
possessed similar energy levels (2.3 kcalÆmol)1) Due to the
different rotamer of the iron-fixing Glu376 in TH, the
oxygen atom trans to His331 was slightly pushed aside
(0.68 A˚)
In contrast to the position ofL-DOPA or DA in PAH
and DA in TH, only one predicted placement ofL-DOPA in
THwas compatible with a bidentate binding mode (Fig 5)
However, the total energy of this conformation was
8.5 kcalÆmol)1 This is about 6.0 kcalÆmol)1 higher than
the total energy of the most likely, monodentate
conforma-tion among the 33 placements ofL-DOPA with a distance
between the catechol moiety and the iron atom of less than
5.0 A˚ Bidentate binding ofL-DOPA to the active site iron
was prevented by electrostatic forces: theL-DOPA
carb-oxylate group was repelled by the negatively charged TH
residue Asp425 In PAH, the residue corresponding to TH
Asp425 is a neutral Val379 so that the charged carboxylate
group of L-DOPA does not interfere with the tight,
bidentate binding of the catechol moiety to the active site
iron
In the catalytic domains of both amino acid hydroxylases,
the positions of L-DOPA and DA overlapped with the
binding site of BH4 This was true for both orientations of
the pterin cosubstrate in TH Consequently, based on the
results from our molecular docking experiments, it can be
predicted that the two catecholamines compete with BH4
for binding to the active site OnceL-DOPA or DA have
formed a chelate complex with the catalytic iron, enzyme
function must be significantly impaired due to the restricted
access of the essential cosubstrate BH4
Discussion
The aim of the present study was to model critical steps
during substrate binding, catalysis and feedback inhibition
of PAHand THby molecular docking The computational
approach allowed first, the creation of binary complexes of
the natural pterin cosubstrate BH4and the catalytic domain
of the enzymes, followed by the docking of amino acid
substrates We thereby mimicked the highly ordered
sequence of substrate binding in TH[6] that was recently
also proposed for PAH[22]
Molecular docking of BH4into the catalytic domain of
PAHresulted in a conformation corresponding to the
position and orientation of 7,8-BH2 in PAHdetermined
by NMR spectroscopy [23] and X-ray crystallography
[14] The distance between the C4a-Atom of BH4and the
iron was 5.97 A˚, thus closer to the value of 6.06 A˚ in the
crystal structure [14] than to the distance of 4.3 A˚
measured by NMR spectroscopy [23] Exactly the same
distance was found for BH4in the recent crystallographic
study of Andersen et al [22] Three H-bonds fixed the
cosubstrate to the protein The N3-bound proton was at
3.71 A˚ from the carboxylate-group of Glu286, so that
additional water-mediated H-bonds might anchor the
guanidinium moiety in the binding pocket [22] The BH4
sidechain made hydrophobic contacts to Ala322 and
Tyr325, two PAHresidues that were recently associated
with mutations in hyperphenylalaninemia or
phenol-ketonuria, respectively [34,35]
The best placement of BH4 in THin our experiments differed from the reported X-ray crystallographic structure
of 7,8-BH2 bound to TH[13] by a 180 rotation of BH4 around its C4a–C8a bond The natural pterin cosubstrate was anchored by two H-bonds and several hydrophobic interactions, which included p-stacking with Phe300 The distance between the metal atom of THand the pterin carbon C4a that is hydroxylated during the enzymatic reaction, was 4.2 A˚, well below 5.6 A˚ as measured in the X-ray crystallography study [13] On the contrary, the conformer calculated byFLEXXwas similar to the orienta-tion of cosubstrate analogs including 7,8-BH2bound to a recombinant, cobalt(II)-substituted human THisoform 1, examined by proton NMR spectroscopy [36] In this study, the distance between C4a of the pterin analog and the iron atom has been measured as 3.7 A˚ Our coordinates of the pterin position are also in agreement with a recent study using the programDOCK4.0 [37] to model the conformation
of BH4and a series of pterin analogs placed into the crystal structure of the THcatalytic domain [15] On the other hand, equivalent energy scores were obtained when BH4 was manually docked into THaccording to the orientation determined by X-ray crystallography [13] Consequently, our results support the conception that in fact two alternative orientations of the pterin cosubstrate exist within the binding site of TH[13–15] The two conformers apparently possess a similar total energy but seem to be differentially favored depending on the experimental con-ditions
Almost identical positions were obtained for phenylala-nine docked into PAHÆBH4or THÆBH4and for the second-ranking conformation of tyrosine docked into THÆBH4, which probably represents the productive position In this position, the carboxylate moiety of the amino acids was anchored by Arg270 in PAHor Arg316 in TH, respectively The pterin orientation in THdid not have a major effect on the position of the amino acid substrate However, a stabilizing hydrogen bond was formed between the tyrosine hydroxyl moiety at position C4 and BH4in the complex with THwhen the pterin cosubstrate was oriented following the crystallographic coordinates of 7,8-BH2 in TH[13] Site-directed mutagenesis of recombinant rat THhas previously demonstrated the critical significance of the salt bridge formed between the carboxylate group of the amino acid substrate and the guanidinium moiety of Arg316 A replacement of Arg316 with lysine was associated with an increase of KTyrby a factor of at least 400 compared with the wild type [20] As Arg316 forms another buried salt bridge to Asp328, replacing Asp328 with serine might render the guanidinium group of Arg316 more mobile As a result, binding of tyrosine to the catalytic site would become less stable, explaining the increase of KTyrby a factor of 60 in the mutant enzyme [20] This substrate position calculated by FLEXXis in agreement with a recent proton NMR spectro-scopic study of a complex consisting of dimeric human PAH (residues Gly103 to Gln428), 7,8-BH2 and phenylalanine [23] As stated in the latter study, the distance between the hydroxylation site of phenylalanine, the C4 carbon atom, and the catalytic iron is 4.34 A˚ [23] This is in good fit with 4.62 A˚ determined byFLEXX for the common position of phenylalanine in the complex with PAHor TH The distance appears optimal for accepting an iron bound oxygen
Trang 8The energetically most favorable placement of tyrosine in
PAHÆBH4 and surprisingly, also in THÆBH4differed
sub-stantially from this common position of the amino acid
substrates Here, the hydroxyl group of tyrosine was
coordinated towards the active-site iron as expected, but
tyrosine formed a hydrogen bond between its ammonium
group and Pro279 in PAHor Asp425 in TH Our data
suggest that in TH, electrostatic attraction of the tyrosine
ammonium moiety by the negatively charged Asp425
(distance 3.4 A˚) and the formation of an H-bond counteract
the repulsion of the tyrosine carboxylate moiety by the same
protein residue (distance 5.3 A˚) Anchored in this position,
tyrosine interferes with the hydroxylation of BH4at carbon
C4a, which is required for catalysis The orientation of BH4
in PAHand in THimplies that dioxygen must be bound
transto His290 or His336, respectively, in order to obtain
access to the pterin C4a The only alternative position of
dioxygen would be trans to Glu330 in PAHor Glu376 in
TH However, the distance between dioxygen in this
position and the pterin C4a would be approximately 5 A˚,
hence incompatible with hydroxylation In addition, the
coordination of the aromatic ring of tyrosine gives it an
unfavorable position for accepting an electrophile Daubner
et al [21] have demonstrated by combined mutations of
PAHthat the replacement with aspartate of residue Val379,
which corresponds to THresidue Asp425, provides only
very low rates ofL-DOPA formation compared with TH
On the other hand, according to the experimentally
established sequence of substrate binding in TH[6], the
binding of dioxygen prior to the amino acid will promote
the placement of tyrosine in the second-ranking, but
productive position
While the unproductive orientation of tyrosine to the
catalytic domain of PAHsufficiently explains the specificity
of this enzyme for its native substrate phenylalanine,
additional factors outside the catalytic domain may be
relevant, too It has been shown that the substrate specificity
of PAHand THis enhanced though not determined by
mechanisms involving the N-terminal regulatory domain
[19] Our models were restricted to the catalytical domains
of PAHand THso that regulatory changes in the
N-terminal regions were not investigated It is conceivable
that phenylalanine exerts an allosteric effect on PAHafter
binding to the regulatory domain [33] The assumption of a
fixed active-site crevice structure precludes the observation
of conformational alterations upon phenylalanine binding
as recently described by Andersen et al [22] In this study,
the placement of tyrosine in PAHwas modeled after
crystallographic coordinates of the phenylalanine analog
3-(2-thienyl)-L-alanine bound to BH4ÆPAH Preserving in
their model both the position of the main chain and the
orientation of the ring structure, the authors concluded that
tyrosine is not accepted by PAHas amino acid substrate
because its hydroxyl oxygen is sterically hindered by the side
chain of Trp326 [22] The sterical interference of tyrosine in
this primary substrate position will certainly be important
for its release from the active site as product after
phenylalanine hydroxylation According to the present
results, however, the preconditions of this model appear
too rigid if one considers tyrosine as an independent ligand
or possible alternative substrate of PAH In this case,
the most probable position of tyrosine in PAH, defined by
the total energy, differs essentially from the position of the native substrate phenylalanine Whether binding of tyrosine
in this position triggers a large change in the protein structure as shown after binding of 3-(2-thienyl)-L-alanine [22] needs to be investigated
Feedback inhibition by catecholamine end products is a major regulatory factor both for PAHand TH Molecular docking of L-DOPA or DA into the crystal structure of either amino acid hydroxylase provided a plausible and energetically favorable conformation of the complex with direct binding of the catecholamine inhibitors to the active-site iron Bidentate binding of the THiron by the two hydroxyl groups of the DA catechol moiety has been suggested based on studies using resonance Raman spectro-scopy [38], or a combination of electron paramagnetic resonance (EPR), extended X-ray absorption fine structure (EXAFS) and Mo¨ssbauer spectroscopy [16] This binding mode was later demonstrated by X-ray crystallography in a binary complex of truncated PAHwith bound catechol-aminesL-DOPA, DA, noradrenaline and adrenaline [17] The position of the amine group of the catecholamine inhibitors is less well defined According to our results, the amine groups ofL-DOPA and DA freely stick out of the active-site pocket in both THand PAH Two distinct conformers ofL-DOPA and DA in the complex with PAH were assigned comparably favorable energy scores The conformers differed by 180 rotation around an imaginary iron-catecholamine axis that runs between the two hydroxyl groups of the catechol moiety In agreement with a previous X-ray crystallographic investigation of binary PAHÆcate-cholamine complexes [17], neither of the two conformers was preferred
We found the same ambiguous binding pattern for DA in
TH, but not forL-DOPA Instead, the predicted position of
L-DOPA in the bidentate binding mode was assigned a high energy This is attributable to the electrostatic repulsion of its negatively charged carboxylate group by THresidue Asp425, whereas in PAH, the corresponding residue represents neutral Val379 A monodentate, therefore less tight and also less stable binding ofL-DOPA to the catalytic iron was predicted by FLEXX Consequently, direct inhibi-tion of THby its native product L-DOPA would be impeded compared with DA In vitro experiments have shown that concentrations of L-DOPA between 10- and 15-fold higher than DA are necessary to inhibit by 50% recombinant human THisoforms 1 and 2 [39] Our findings suggest that in TH, electrostatic forces play a key role in hindering an immediate interference of the reaction product with the catalytic center The negative charge of the corresponding THresidue Asp425 is a limiting factor for the access ofL-DOPA to the catalytic iron, thereby reducing product inhibition
As the binding sites of catecholamines and BH4overlap
in both amino acid hydroxylases, they compete with each other for obtaining access to the catalytic site Moreover, bidentate binding of catecholamines to the catalytic iron(II) causes its oxidation to the ferric form Stoichiometric amounts of DA rapidly induce iron oxidation and enzyme inactivation [40] Due to the stability of the generated complex, catecholamines turn into almost irreversible inhibitors [3,8] Considering that catecholamines are redu-cing agents, oxidation of the catalytic iron provoked by the
Trang 9binding of these inhibitors must surprise We hypothesize
that after binding to the iron atom, catecholamines activate
dioxygen in analogy to the activation of dioxygen by BH4
during catalysis According to our hypothesis, a highly
reactive iron-oxo intermediate would form together with the
generation of DA quinone and a hydroxyl anion (Fig 6)
As the remaining iron-bound oxygen atom is now
neigh-boring solvent water molecules instead of an electron-rich
nucleophile such as the aromatic ring in tyrosine or
phenylalanine, the intermediate is likely to decompose into
oxidized iron and a highly reactive hydroxyl radical (Fig 6)
The generation of reactive oxygen species during in vitro
tyrosine hydroxylation has been reported previously,
although it was attributed to partial uncoupling of BH4
oxidation during catalysis [41] In our model, the generation
of reactive oxygen species depends on the stoichiometric
equilibrium of the aromatic amino acid hydroxylase, the
cosubstrate BH4 and catecholamine end products A
clinically important shift in this equilibrium may occur in
Parkinson’s disease, as patients are systemically treated with
L-DOPA, which is intracerebrally decarboxylized to DA
Post mortem investigations of parkinsonian brains and
animal studies have shown that degenerating dopaminergic
neurons in the substantia nigra are specifically vulnerable to
reactive oxygen species due to a reduction in their
antioxi-dative defense systems such as glutathion [42] As an
unwanted side-effect, high doses of -DOPA may add to the
oxidative stress by tipping the balance towards THinhibi-tion and iron oxidaTHinhibi-tion
Acknowledgment
The authors wish to thank Marcus Gastreich, Jannis Apostolakis, Joachim Selbig and Volker Schu¨nemann for constructive discussions and helpful comments on the manuscript This research was supported
in part by the Faculty of Medicine of the Medical University of Lu¨beck (MUL J031).
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