Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase Astrid Maaß 1 , Joachim Scholz 2,3 and Andreas Moser 2 1 Fraunhofer-Institute for Algorithms and Scientific Computing (SCAI), Schloss Birlinghoven, Sankt Augustin, Germany; 2 Neurochemistry Research Group, Department of Neurology, Medical University of Lu ¨ beck, Lu ¨ beck, Germany; 3 Neural Plasticity Research 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 PAH and 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 PAH and 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 (BH 4 ) and the substrates phenylalanine and tyrosine to the catalytic domains of PAH and TH. The amino acid substrates were placed in positions common to both enzymes. The productive position of tyrosine in THÆBH 4 was stabilized by a hydrogen bond with BH 4 . Despite favorable energy scores, tyrosine in a position trans to PAH residue His290 or TH residue 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 and L -DOPA in PAH and for DA in TH. Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction product L -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 TH and PAH possess 65% sequence identity and 80% homology [3] (Fig. 1). In a catalytic mechanism shared by PAH and 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 (BH 4 ) are essential cosubstrates of the reaction. A coupled hydroxylation of the amino acid and the pterin takes place after all three substrates (BH 4 , dioxygen, amino acid) have bound to the active site [6,7]. TH and PAH are 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. BH 4 and lead to oxidation of the catalytic iron [8,9]. Analyses of truncated forms of rat TH, rat and human PAH by 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-BH 2 ) bound to truncated rat TH and human PAH has 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 TH activity 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) Eur. J. Biochem. 270, 1065–1075 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03429.x TH and crystallographic data obtained from binary com- plexes of catecholamines and truncated human PAH have demonstrated that the two hydroxyl groups of the catechol moiety bind to the catalytic iron [16–18]. Despite the similarities between TH and PAH regarding thestructureoftheactivesiteandthecatalyticmecha- nism, there is one striking difference: TH accepts also phenylalanine as substrate, with K m increased by a factor of six and V max decreased by a factor of four compared with tyrosine [19]. In contrast, PAH is 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 PAH and 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 PAH and 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 PAH with 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 PAH and TH has been used to model the full length structure of TPH [24]. We have modeled the catalytic sites of PAH and TH and introduced BH 4 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 PAH and TH by catecholamine end products to explain the reduced susceptibility of TH to feedback inhibition by L -DOPA compared with DA. Experimental procedures Ligand–protein complexes were generated based on the crystal structures 5pah for PAH and 2toh for TH (Protein Data Bank) [13,17]. The software FLEXX , version 1.7.6 was used for ligand docking [25]. The complexes were optimized by force-field energy minimization using CHARMM ,version 23.2 [26]. CHARMM and CAMLAB , 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-BH 2 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. TH residue 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 e ¼ 2r and convergence ensured throughout. Ligand structures were divided into fragments and reconstructed stepwise within the active site using FLEXX . 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 (e ¼ 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 the CHARMM force field energy in a homogeneous dielectric medium (e ¼ 4) plus the solvation energy calculated by CAMLAB . 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 e internal ¼ 4 inside the molecular surface of the complex, and e external ¼ e H2O ¼ 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 e internal ¼ e external ¼ 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. 1066 A. Maaß et al. (Eur. J. Biochem. 270) Ó FEBS 2003 energies. The total electric charge was +6 for 5pah and )16 for 2toh, as 2toh comprises the catalytic core domain and the tetramerization domain. BH 4 is uncharged. The aroma- tic amino acids phenylalanine and tyrosine were implemen- ted in their zwitterionic state with a protonated 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 TH and PAH with bound 7,8-BH 2 (2toh, 1dmw), the carbonyl oxygen of 7,8-BH 2 is the atom closest to the active site iron with a distance of 3.6 or 3.8 A ˚ , respectively. Therefore, conformations of BH 4 with a maximal distance d BH 4 -Fe of 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 d Tyr-Fe or d Phe-Fe between 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 d L -DOPA-Fe or d DA-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 BH 4 into the crystal structure of the PAH catalytic 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 PAH catalytic iron atom (d BH 4 -Fe ) waslessthan4.5A ˚ (Fig. 3). The energetically most favorable conformer corresponded with the position of 7,8-BH 2 bound to PAH at 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 PAH residue 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 BH 4 into the crystal structure of TH produced 300 conformations; in 46 out of these, the distance d BH 4 -Fe was shorter than 4.5 A ˚ (Fig. 3). The energetically most favorable placement was very similar to the conformation of BH 4 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 BH 4 coordinated 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 BH 4 coincided with the conformation of BH 4 in TH previously computed by Alma ˚ s et al.using DOCK 4.0 (rmsd 2.2 ± 0.2 A ˚ ) [15]. However, it differed from the conformation of 7,8-BH 2 in the binary complex identified by X-ray crystallography [13]. In the latter study, the position of 7,8-BH 2 was rotated by 180° and characterized by a p-stacking interaction of the planar pterin moiety with TH residue Phe300, tilted by 10°. We manually docked BH 4 into TH to further investigate this alternative pterin position. The energetically most favorable manual placement was in good agreement with the rotated position of 7,8-BH 2 cocrystallized 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 BH 4 formed an H-bond to the carboxylate group of Glu332. The distance d BH 4 -Fe was 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 )1 compared with 1.5 kcalÆmol )1 for 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. Ó FEBS 2003 Modeled ligand–protein complexes of PAH and TH (Eur. J. Biochem. 270) 1067 presumably artificial straining of the deeply buried BH 4 - 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 PAHÆBH 4 and THÆBH 4 After docking the native substrate phenylalanine into PAHÆBH 4 , 73 candidate positions with a distance d Phe-Fe between the center of the aromatic ring and the TH iron 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 PAHÆBH 4 . This phenyl- alanine position provided by FLEXX and the calculated hydrogen bonds to surrounding PAH residues 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-BH 2 that was previously calculated after restraints from NMR spectroscopy [23] (rmsd 2.09 A ˚ and 1.29 A ˚ , respectively). Docking of tyrosine into PAHÆBH 4 produced a set of 179 conformations. Out of these, 55 conformations fulfilled the distance criterion of d Tyr-Fe being 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 PAH differed significantly from that of phenylalanine. FLEXX provided 318 possible conformations of tyrosine in THÆBH 4 . 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 PAH is shown in red, for BH 4 in TH according to the results of the automated docking in blue, and for BH 4 in TH according to the manual docking in the X-ray crystallographic mode in green. Hydrogens are omitted for clarity. 1068 A. Maaß et al. (Eur. J. Biochem. 270) Ó FEBS 2003 was 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 PAHÆBH 4 , the hydroxyl group was placed trans to TH residue 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 PAHÆBH 4 however, FLEXX provided a second conformation for tyrosine, which was almost identical to the position predicted for the native substrate phenylalanine in the complex with PAHÆBH 4 . 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 TH residues Ser324 and Pro325 (Fig. 4). The orientation of the pterin cosubstrate in the complex with TH did not have an effect on the position of tyrosine. However, the orientation of BH 4 in the second- ranking conformation of tyrosine in THÆBH 4 corresponded to that of 7,8-BH 2 cocrystallized with TH [13] and in this orientation, N3 of BH 4 exhibited a stabilizing H-bond to the tyrosine hydroxyl group. The best placement of phenylalanine in THÆBH 4 coincided with the second-ranking position of tyrosine, and it also corresponded to the position obtained by phenylalanine in PAHÆBH 4 (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 THÆBH 4 (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 . PAH residue Phenylalanine in PAHÆBH 4 Tyrosine in PAHÆBH 4 E vdW E Coulomb H-bonds PAH residue E vdW E Coulomb H-bonds 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 Pro279 – 0.50 0.23 His385 – 1.13 0.48 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 TH with 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 TH is given in light green. BH 4 in the corresponding complexes is shown in the same color as the amino acid substrate. Ó FEBS 2003 Modeled ligand–protein complexes of PAH and TH (Eur. J. Biochem. 270) 1069 Differences in the binding of L -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 products L -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. FLEXX yielded 189 conformations of L -DOPA and 213 conformations of DA in 5pah; 32 candidate positions for L -DOPA and 55 positions for DA were within a distance of 5.0 A ˚ .Nine independent predictions for L -DOPA converged to the same local minimum, which was characterized by an H-bond between the amine group of L -DOPA and the backbone oxygen of PAH residue 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). Thedifferenceof5.5kcalÆmol )1 in 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 PAH two equivalent positions exist for L -DOPA and DA, with the plane of the catechol ring rotated 180°. The docking of DA into TH yielded 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 TH residue 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. 1070 A. Maaß et al. (Eur. J. Biochem. 270) Ó FEBS 2003 2.15 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 of L -DOPAorDAinPAH and DA in TH, only one predicted placement of L -DOPA in TH was 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 of L -DOPA with a distance between the catechol moiety and the iron atom of less than 5.0 A ˚ . Bidentate binding of L -DOPA to the active site iron was prevented by electrostatic forces: the L -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 BH 4 . 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 BH 4 for binding to the active site. Once L -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 BH 4 . Discussion The aim of the present study was to model critical steps during substrate binding, catalysis and feedback inhibition of PAH and TH by molecular docking. The computational approach allowed first, the creation of binary complexes of the natural pterin cosubstrate BH 4 and 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 BH 4 into the catalytic domain of PAH resulted in a conformation corresponding to the position and orientation of 7,8-BH 2 in PAH determined by NMR spectroscopy [23] and X-ray crystallography [14]. The distance between the C4a-Atom of BH 4 and 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 BH 4 in 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 BH 4 sidechain made hydrophobic contacts to Ala322 and Tyr325, two PAH residues that were recently associated with mutations in hyperphenylalaninemia or phenol- ketonuria, respectively [34,35]. ThebestplacementofBH 4 in TH in our experiments differed from the reported X-ray crystallographic structure of 7,8-BH 2 bound to TH [13] by a 180° rotation of BH 4 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 TH and the pterin carbon C4a that is hydroxylated during the enzymatic reaction, was 4.2 A ˚ ,wellbelow5.6A ˚ as measured in the X-ray crystallography study [13]. On the contrary, the conformer calculated by FLEXX was similar to the orienta- tion of cosubstrate analogs including 7,8-BH 2 bound to a recombinant, cobalt(II)-substituted human TH isoform 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 program DOCK 4.0 [37] to model the conformation of BH 4 and a series of pterin analogs placed into the crystal structure of the TH catalytic domain [15]. On the other hand, equivalent energy scores were obtained when BH 4 was manually docked into TH according 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 PAHÆBH 4 or THÆBH 4 and for the second- ranking conformation of tyrosine docked into THÆBH 4 , which probably represents the productive position. In this position, the carboxylate moiety of the amino acids was anchored by Arg270 in PAH or Arg316 in TH, respectively. The pterin orientation in TH did 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 BH 4 in the complex with TH when the pterin cosubstrate was oriented following the crystallographic coordinates of 7,8-BH 2 in TH [13]. Site- directed mutagenesis of recombinant rat TH has 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 K Tyr by 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 K Tyr byafactorof60inthe mutant enzyme [20]. This substrate position calculated by FLEXX is in agreement with a recent proton NMR spectro- scopic study of a complex consisting of dimeric human PAH (residues Gly103 to Gln428), 7,8-BH 2 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 by FLEX X for the common position of phenylalanine in the complex with PAH or TH. The distance appears optimal for accepting an iron bound oxygen. Ó FEBS 2003 Modeled ligand–protein complexes of PAH and TH (Eur. J. Biochem. 270) 1071 The energetically most favorable placement of tyrosine in PAHÆBH 4 and surprisingly, also in THÆBH 4 differed 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 PAH or 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 BH 4 at carbon C4a, which is required for catalysis. The orientation of BH 4 in PAH and in TH implies that dioxygen must be bound trans to 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 PAH or 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 PAH that the replacement with aspartate of residue Val379, which corresponds to TH residue Asp425, provides only very low rates of L -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 PAH sufficiently 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 PAH and TH is enhanced though not determined by mechanisms involving the N-terminal regulatory domain [19]. Our models were restricted to the catalytical domains of PAH and TH so that regulatory changes in the N-terminal regions were not investigated. It is conceivable that phenylalanine exerts an allosteric effect on PAH after 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 PAH was modeled after crystallographic coordinates of the phenylalanine analog 3-(2-thienyl)- L -alanine bound to BH 4 ÆPAH. 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 PAH as 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 PAH and 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 TH iron 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 PAH with bound catechol- amines L -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 of L -DOPA and DA freely stick out of the active-site pocket in both TH and PAH. Two distinct conformers of L -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 for L -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 TH residue Asp425, whereas in PAH, the corresponding residue represents neutral Val379. A monodentate, therefore less tight and also less stable binding of L -DOPA to the catalytic iron was predicted by F LEX X. Consequently, direct inhibi- tion of TH by 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 TH isoforms 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 TH residue Asp425 is a limiting factor for the access of L -DOPA to the catalytic iron, thereby reducing product inhibition. As the binding sites of catecholamines and BH 4 overlap 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 1072 A. Maaß et al. (Eur. J. Biochem. 270) Ó FEBS 2003 binding 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 BH 4 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 BH 4 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 BH 4 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 L -DOPA may add to the oxidative stress by tipping the balance towards TH inhibi- tion and iron oxidation. 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). References 1. Kappock, T.J. & Caradonna, J.P. (1996) Pterin-dependent amino acid hydroxylases. Chem. Rev. 96, 2659–2756. 2. Fitzpatrick, P.F. (1999) Tetrahydropterin-dependent amino acid hydroxylases. Ann. Rev. Biochem. 68, 355–381. 3. Flatmark, T. & Stevens, R.C. (1999) Structural insight into the aromatic amino acid hydroxylases and their disease-related mutant forms. Chem. Rev. 99, 2137–2160. 4. Walker, S.J., Liu, X., Roskoski, R. & Vrana, K.E. (1994) Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of bacterially expressed enzyme. Biochim. Biophys. Acta 1206, 113– 119. 5. Dickson, P.W., Jennings, I.G. & Cotton, R.G.H. (1994) Deli- neation of the catalytic core of phenylalanine hydroxylase and identification of glutamate 286 as a critical residue for pterin function. J. Biol. Chem. 269, 20369–20375. Fig. 6. Hydroxylation of tyrosine and a possible sequence of events related to the feedback inhibition of TH by the representative catecholamine DA. The substrates of TH bind in a highly ordered sequence, starting with (1) BH 4 followed by (2) dioxygen and (3) the amino acid substrate tyrosine. (4) During the catalytic step, one oxygen atom reacts with BH 4 to produce 4a-OH- BH 4 . The second oxygen atom forms an iron-oxo-intermediate before the actual hydroxylation of the tyrosine aromatic ring takes place. (5) After its release from the catalytic site, (6) L -DOPA is decarboxylated to DA by the aromatic L -amino acid decarboxylase (EC 4.1.1.28). (7) DA can bind to the TH catalytic iron via the hydroxyl groups of its catechol moiety in a bidentate fashion. (8) DA activates dioxygen in analogy to the catalytic activation of dioxygen by BH 4 during catalysis. (9) As a result, a highly reactive iron-oxo intermediate is generated. (10) In the following reaction step, DA quinone and a hydroxyl anion are formed. In the presence of solvent water and due to the lack of an electron-rich nucleophile, the intermediate decomposes into oxidized iron and a highly reactive hydroxyl radical. (11) BH 4 is required to reduce the ferric iron and re-establish TH activity. Ó FEBS 2003 Modeled ligand–protein complexes of PAH and TH (Eur. J. Biochem. 270) 1073 6. Fitzpatrick, P.F. (1991) Steady-state kinetic mechanism of rat tyrosine hydroxylase. Biochemistry 30, 3658–3662. 7. Fitzpatrick, P.F. (1991) Studies of the rate-limiting step in the tyrosine hydroxylase reaction: alternate substrates, solvent isotope effects, and transition-state analogues. Biochemistry 30, 6386– 6391. 8. Kumer, S.C. & Vrana, K.E. (1996) Intricate regulation of tyrosine hydroxylase activity and gene expression. J. Neurochem. 67, 443– 462. 9. Ramsey, A.J. & Fitzpatrick, P.F. (2000) Effects of phosphoryl- ation on binding of catecholamines to tyrosine hydroxylase: spe- cificity and thermodynamics. Biochemistry 39, 773–778. 10. Erlandsen, H., Fusetti, F., Martinez, A., Hough, E., Flatmark, T. & Stevens, R.C. (1997) Crystal structure of the catalytic domain of human phenylalanine hydroxylase reveals the structural basis for phenylketonuria. Nat. Struct. Biol. 4, 995–1000. 11. Goodwill, K.E., Sabatier, C., Marks, C., Raag, R., Fitzpatrick, P.F. & Stevens, R. (1997) Crystal structure of tyrosine hydroxylase at 2.3 A ˚ and its implications for inherited neurodegenerative dis- eases. Nat. Struct. Biol. 4, 578–585. 12. Fusetti, F., Erlandsen, H., Flatmark, T. & Stevens, R.C. (1998) Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria. J. Biol. Chem. 237, 16962– 16967. 13. Goodwill, K.E., Sabatier, C. & Stevens, R.C. (1998) Crystal structure of tyrosine hydroxylase with bound cofactor analogue andironat2.3A ˚ resolution: self-hydroxylation of Phe300 and the pterin-binding site. Biochemistry 37, 13437–13445. 14. Erlandsen, H., Flatmark, T. & Stevens, R.C. (2000) Crystal structure and site-specific mutagenesis of pterin-bound human phenylalanine hydroxylase. Biochemistry 39, 2208–2217. 15. Alma ˚ s, B., Toska, K., Teigen, K., Groehn, V., Pfleiderer, W., Martinez, A., Flatmark, T. & Haavik, J. (2000) A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase. Biochemistry 39, 13676–13686. 16. Meyer-Klaucke,W.,Winkler,H.,Trautwein,A.X.,Nolting,H.F. & Haavik, J. (1996) Mo ¨ ssbauer, electron-paramagnetic-resonance and X-ray-absorption fine-structure studies of the iron environ- ment in recombinant human tyrosine hydroxylase. Eur. J. Bio- chem. 241, 432–439. 17. Erlandsen, H., Flatmark, T., Stevens, R.C. & Hough, E. (1998) Crystallographic analysis of the human phenylalanine hydroxylase catalytic domain with bound catechol inhibitors at 2.0 A ˚ resolu- tion. Biochemistry 37, 15638–15646. 18. Schu ¨ nemann, V., Meier, C., Meyer-Klaucke, W., Winkler, H., Trautwein, A.X., Knappskog, P.M., Toska, K. & Haavik, J. (1999) Iron coordination geometry in full-length, truncated, and dehydrated forms of human tyrosine hydroxylase studied by Mo ¨ ssbauer and X-ray absorption spectroscopy. J. Biol. Inorg. Chem. 4, 223–231. 19. Daubner, S.C., Hillas, P.J. & Fitzpatrick, P.F. (1997) Characteri- zation of chimeric pterin-dependent hydroxylases: contributions of the regulatory domains of tyrosine and phenylalanine hydro- xylase to substrate specificity. Biochemistry 36, 11574–11582. 20. Daubner, S.C. & Fitzpatrick, P. (1999) Site-directed mutants of charged residues in the active site of tyrosine hydroxylase. Biochemistry 38, 4448–4454. 21. Daubner, S.C., Melendez, J. & Fitzpatrick, P. (2000) Reversing the substrate specificities of phenylalanine and tyrosine hydroxylase: aspartate 425 of tyrosine hydroxylase is essential for 1-DOPA formation. Biochemistry 39, 9652–9661. 22. Andersen, O.A., Flatmark, T. & Hough, E. (2002) Crystal struc- ture of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2- thienyl)- L -alanine, and its implications for the mechanism of catalysis and substrate activation. J. Mol. Biol. 320, 1095–1108. 23. Teigen, K., Frøystein, N. & Martinez, A. (1999) The structural basis of the recognition of phenylalanine and pterin cofactors by phenylalanine hydroxylase: implications for the catalytic mechanism. J. Mol. Biol. 294, 807–823. 24. Jiang, G.C T., Yohrling, G.J., Schmitt, I.V.J.D. & Vrana, K.E. (2000) Identification of substrate orienting and phosphorylation sites within tryptophan hydroxylase using homology-based molecular modeling. J. Mol. Biol. 302, 1005–1017. 25. Rarey, M., Kramer, B., Lengauer, T. & Klebe, G. (1996) A fast flexible docking method using an incremental construction algo- rithm. J. Mol. Biol. 261, 470–489. 26. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S. & Karplus, M. (1983) CHARMm: a program for macromolecular energy, minimization and dynamics calcula- tion. J. Comp. Chem. 4, 187–217. 27. Hoffmann, D., Kramer, B., Washio, T., Steinmetzer, T., Rarey, M. & Lengauer, T. (1999) Two-stage method for protein-ligand docking. J. Med. Chem. 42, 4422–4433. 28. Ellis, H.R., Daubner, S.C., McCulloch, R.I. & Fitzpatrick, P.F. (1999) Phenylalanine residues in the active site of tyrosine hydroxylase: mutagenesis of Phe300 and Phe309 to alanine and metal ion-catalyzed hydroxylation of Phe300. Biochemistry 38, 10909–10914. 29. Bohm, H J. (1994) The development of a simple empirical scoring function to estimate the binding constant for a protein-ligand complex of known three-dimensional structure. J. Comput. Aided Mol. Des. 8, 243–256. 30. Warwicker, J. & Watson, H.C. (1982) Calculation of the electric potential in the active site cleft due to alpha-helix dipoles. J. Mol. Biol. 157, 671–679. 31. Klapper, I., Hagstrom, R., Fine, R. & Honig, B. (1986) Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins 1, 47–59. 32. Hoffmann,D.,Washio,T.,Gessler,K.&Jacob,J.(1998)Tackling concrete problems in molecular biophysics using Monte Carlo and related methods: glycosylation, folding, solvation. In: Proceedings of the Workshop on: Monte Carlo Approach to Biopolymers and Protein Folding (Grassberger, P., Barkema, G. & Nadler, W., eds), pp. 153–170. World Scientific Publishing, River Edge, Singapore. 33. Kobe, B., Jennings, I.G., House, C.M., Michell, B.J., Goodwill, K.E., Santarsiero, B.D., Stevens, R.C., Cotton, R.G.H. & Kemp, B.C. (1999) Structural basis of autoregulation of phenylalanine hydroxylase. Nat. Struct. Biol. 6, 442–448. 34. Svensson, E., Eisensmith, R.C., Dworniczak, B., van Dobeln, U., Hagenfeld, L., Horst, J. & Woo, S.L. (1992) Two missense mutations causing mild hyperphenylalaninemia associated with DNA haplotype 12. Hum. Mutat. 1, 129–137. 35. Nowacki, P., Byck, S., Prevost, L. & Scriver, C.R. (1998) PAH Mutation Analysis Consortium Database: 1997. Prototype for relational locus-specific mutation databases. Nucleic Acids Res. 26, 220–225. 36. Martinez, A., Vageli, O., Pfleiderer, W. & Flatmark, T. (1998) Proton NMR studies on the conformation of the pterin cofactor bound at the active site of recombinant human tyrosine hydro- xylase. Pteridines 9, 44–52. 37. Meng, E.C., Schichet, B.K. & Kuntz, I.D. (1992) Automated docking with grid-based energy evaluation. J. Comput. Chem. 13, 505–524. 38. Michaud-Soret, I., Andersson, K.K., Que, L. Jr & Haavik, J. (1995) Resonance Raman studies of catecholate and phenolate complexes of recombinant human tyrosine hydroxylase. Biochemistry 34, 5504–5510. 39. Alma ˚ s, B., Le Bourdelles, B., Flatmark, T., Mallet, J. & Haavik, J. (1992) Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. 1074 A. Maaß et al. (Eur. J. Biochem. 270) Ó FEBS 2003 [...]...Ó FEBS 2003 Modeled ligand–protein complexes of PAH and TH (Eur J Biochem 270) 1075 Structure/activity studies and mechanistic implications Eur J Biochem 209, 249–255 40 Haavik, J., Martinez, A., Olafsdottir, S., Mallet, J & Flatmark, T (1992) The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy... Flatmark, T (1997) Generation of reactive oxygen species by tyrosine hydroxylase: a possible contribution to the degeneration of dopaminergic neurons? J Neurochem 68, 328–332 42 Blum, D., Torch, S., Lambeng, N., Nissou, M., Benabid, A.L., Sadoul, R & Verna, J.M (2001) Molecular pathways involved in the neurotoxicity of 6-OHDA, dopamine and MPTP: contribution to the apoptotic theory in Parkinson’s disease . Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase Astrid. binding to the catalytic domains of PAH and TH Docking of the native cosubstrate BH 4 into the crystal structure of the PAH catalytic domain yielded a total of 286 conformations. Out of these, 44. actual features of the structural environment defining the active site underlie the substrate specificity of PAH and TH. Rapid conversion of active cosubstrates and substrates into their products