Endogenous tetrahydroisoquinolines associated with Parkinson’s disease mimic the feedback inhibition of tyrosine hydroxylase by catecholamines Joachim Scholz1,2,*, Karen Toska3,*, Alexander Luborzewski1, Astrid Maass4, Volker Schunemann5, ă Jan Haavik3 and Andreas Moser1 Neurochemistry Research Group, Department of Neurology, University of Lubeck, Germany ă Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA Department of Biomedicine, Section of Biochemistry and Molecular Biology, University of Bergen, Norway Fraunhofer-Institute for Algorithms and Scientific Computing (SCAI), Sankt Augustin, Germany Department of Physics, Technical University Kaiserslautern, Germany Keywords enzyme stability; feedback inhibition; Parkinson’s disease; tetrahydroisoquinolines; tyrosine hydroxylase Correspondence J Scholz, Neural Plasticity Research Group, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Room 4309, Charlestown, MA 02129, USA Fax: +1 617 7243632 Tel: +1 617 7243623 E-mail: scholz.joachim@mgh.harvard.edu *These authors contributed equally to this work (Received 14 November 2007, revised 23 January 2008, accepted 28 February 2008) doi:10.1111/j.1742-4658.2008.06365.x N-methyl-norsalsolinol and related tetrahydroisoquinolines accumulate in the nigrostriatal system of the human brain and are increased in the cerebrospinal fluid of patients with Parkinson’s disease We show here that 6,7-dihydroxylated tetrahydroisoquinolines such as N-methyl-norsalsolinol inhibit tyrosine hydroxylase, the key enzyme in dopamine synthesis, by imitating the mechanisms of catecholamine feedback regulation Docked into a model of the enzyme’s active site, 6,7-dihydroxylated tetrahydroisoquinolines were ligated directly to the iron in the catalytic center, occupying the same position as the catecholamine inhibitor dopamine In this position, the ligands competed with the essential tetrahydropterin cofactor for access to the active site Electron paramagnetic resonance spectroscopy revealed that, like dopamine, 6,7-dihydroxylated tetrahydroisoquinolines rapidly convert the catalytic iron to a ferric (inactive) state Catecholamine binding increases the thermal stability of tyrosine hydroxylase and improves its resistance to proteolysis We observed a similar effect after incubation with N-methyl-norsalsolinol or norsalsolinol Following an initial rapid decline in tyrosine hydroxylation, the residual activity remained stable for h at 37 °C Phosphorylation by protein kinase A facilitates the release of bound catecholamines and is the most prominent mechanism of tyrosine hydroxylase reactivation Protein kinase A also fully restored enzyme activity after incubation with N-methyl-norsalsolinol, demonstrating that tyrosine hydroxylase inhibition by 6,7-dihydroxylated tetrahydroisoquinolines mimics all essential aspects of catecholamine end-product regulation Increased levels of N-methylnorsalsolinol and related tetrahydroisoquinolines are therefore likely to accelerate dopamine depletion in Parkinson’s disease Abbreviations CSF, cerebrospinal fluid; DA, dopamine; hTH, human tyrosine hydroxylase; L-DOPA, L-3,4-dihydroxyphenylalanine; MPTP, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine; NMNorsal, N-methyl-norsalsolinol; NMSal, N-methyl-salsolinol; NMTIQ, N-methyl-1,2,3,4-tetrahydroisoquinoline; Norsal, norsalsolinol; PD, Parkinson’s disease; PKA, protein kinase A; ROS, reactive oxygen species; TH, tyrosine hydroxylase; TIQ, tetrahydroisoquinoline FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS 2109 TIQs imitate mechanisms of TH feedback inhibition J Scholz et al N-methyl-norsalsolinol, salsolinol and N-methyl-salsolinol are endogenous tetrahydroisoquinolines (TIQs) formed through non-enzymatic condensation of dopamine (DA) with aldehydes or pyruvic acid Increased concentrations of these TIQs are found in the cerebrospinal fluid (CSF) of patients with Parkinson’s disease (PD) [1–3] Accumulation of N-methylated TIQs in the substantia nigra and the corpus striatum of the human brain [2] and their structural similarity to 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Fig 1) have led to the hypothesis that TIQs are directly involved in the degeneration of dopaminergic neurons Like MPTP, TIQs inhibit mitochondrial respiration However, the toxicity of TIQs is low because of their limited ability to cross the mitochondrial membrane [4] High concentrations of N-methyl-salsolinol (NMSal) are required to induce apoptosis of dopaminergic cells in vitro [5], and NMSal causes a loss of tyrosine hydroxylase-immunoreactive neurons in the rat substantia nigra in vivo only after repeated stereotaxic injections [6] Some TIQs even have neuroprotective effects [7,8] Rather than provoking neuronal degeneration, endogenous TIQs may interfere with DA synthesis N-methyl-norsalsolinol (NMNorsal) [9] and salsolinol [10,11] inhibit tyrosine hydroxylase (TH; tyrosine 3-monooxygenase, EC 1.14.16.2), the key enzyme in DA synthesis, in vitro, and a single injection of NMSal into the rat corpus striatum markedly reduces TH activity in vivo, leading to an almost com- Fig Chemical structures of DA and the TIQs examined in this study The 6,7-dihydroxylated TIQs Norsal, NMNorsal and NMSal have an intact catechol moiety NMNorsal and NMSal are endogenous compounds with structural similarity to MPTP 2110 plete loss of DA in the absence of neuronal degeneration [6] The CSF levels of TIQs increase in early PD and decrease as the disease progresses [12] TH inhibition by endogenous TIQs may therefore be most prominent at a critical time, when surviving substantia nigra neurons are challenged by the necessity to increase DA synthesis and release in order to uphold the functional integrity of the nigrostriatal pathway [13–15] Such adaptive neurochemical changes are likely to delay the appearance of clinical signs in PD, which lags several years behind the onset of dopaminergic neuron degeneration in the substantia nigra [16] Animal models of PD have demonstrated the plasticity of the nigrostriatal system For example, near-complete recovery of motor function is achieved when striatal DA levels are restored by concomitant virus-mediated transfer of the genes encoding TH and GTP cyclohydrolase, the ratelimiting synthetic enzyme for the essential TH cofactor 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) [17] Thus, understanding how TIQs block TH activity is important in order to develop treatment strategies that help to sustain dopaminergic nigrostriatal signaling in early PD TH catalyzes the hydroxylation of tyrosine to l-3,4dihydroxyphenylalanine (l-DOPA), which is the ratelimiting step in synthesis of the catecholamines DA, norepinephrine and epinephrine TH consists of four identical subunits that contain a C-terminal catalytic domain (residues 156–498) and an N-terminal regula˚ tory domain The active site of the enzyme is a 17 A deep crevice with a ferrous iron atom located in its center [18] Alternative mRNA splicing of a single primary transcript generates at least four isoforms of human TH (hTH) that are differentially expressed in tissues; the most prominent isoforms in the brain are hTH1 and hTH2 The isoforms differ only in the N-terminal regulatory region; the C-terminal domain with the active site is identical in all hTH isoforms, and the catalytic domain is also highly conserved across animal species and in other aromatic amino acid hydroxylases [19] TH activity is subject to intricate regulation Transcriptional control, modulation of RNA stability, translational regulation and enzyme stability establish a steady-state level of TH protein [20,21] Short-term regulation of TH activity includes feedback inhibition by catecholamine end products, allosteric modulation and phosphorylation-dependent activation by various kinases [22,23] Using recombinant hTH1 and hTH4, we have examined the inhibitory effect of NMNorsal and structurally related TIQs on TH Molecular docking revealed that 6,7-dihydroxylated TIQs associated with PD FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS J Scholz et al compete with the essential tetrahydropterin cofactor of hTH for access to the enzyme’s active site, whereas MPTP has low affinity for the amino acid binding site By binding directly to the ferrous iron atom in the catalytic center and converting the iron to a ferric state, 6,7-dihydroxylated TIQs block hTH activity through a mechanism that mimics the physiological feedback inhibition by catecholamines Unlike DA, which caused a near complete loss of hTH activity over time, NMNorsal stabilized hTH, but with a reduced level of activity TIQs imitate mechanisms of TH feedback inhibition is unmethylated; NMTIQ lacks the two hydroxyl residues at positions and of its benzene ring (Fig 1) Norsal decreased enzymatic l-DOPA synthesis with an IC50 of 10.0 lm (Fig 2A) In contrast, hTH activity remained unchanged in the presence of the nonhydroxylated NMTIQ (Fig 2A) Consequently, hTH inhibition depends critically on the catechol moiety of 6,7-dihydroxylated TIQs Methylation of the piperidine nitrogen or a neighboring carbon modulates the efficacy of 6,7-dihydroxylated TIQs in reducing hTH activity, but is not responsible for the overall inhibitory effect Results Molecular docking 6,7-Dihydroxylated TIQs inhibit human TH The recombinant isoforms hTH1 and hTH4 produced 539 ± 41 nmolỈmin)1Ỉmg)1 and 564 ± 40 nmolỈ min)1Ỉmg)1 l-DOPA, respectively Human TH activity decreased in the presence of NMNorsal and NMSal, two 6,7-dihydroxylated TIQs (Fig 1) that have previously been identified in the CSF of patients with PD [1,3,12] NMNorsal inhibited hTH almost as strongly (IC50 = 0.3 lm) as the catecholamine end product DA (IC50 = 0.2 lm), whereas higher concentrations of NMSal (IC50 = 4.0 lm) were required to reduce hTH activity (Fig 2A) A kinetic analysis indicated that NMNorsal reduced hTH activity by competing with the essential pterin cofactor (Fig 2B); hTH inhibition by NMNorsal was noncompetitive with respect to tyrosine (data not shown) We compared the inhibitory effects of NMNorsal and NMSal with those of two other TIQs, norsalsolinol (Norsal) and N-methyl-1,2,3,4-tetrahydroisoquinoline (NMTIQ) Norsal has an intact catechol moiety like NMNorsal and NMSal, but its piperidine nitrogen TH belongs to a family of tetrahydropterin-dependent amino acid hydroxylases that also includes phenylalanine hydroxylase and tryptophan hydroxylase These enzymes are composed of four identical subunits, each containing a divalent iron atom in its catalytic domain that is required for activity [24] To explore the mechanism of TH inhibition by 6,7-dihydroxylated TIQs, we identified potential binding sites of NMNorsal, NMSal and Norsal in the crystal structure of the enzyme’s catalytic domain (Protein Data Bank identification code 2toh) [18] using molecular docking We also determined the energetically favored docking sites for NMTIQ and MPTP, and compared all conformations with the binding site of the physiological feedback inhibitor DA The most favorable placements of NMNorsal, NMSal and Norsal overlapped almost completely and were identical to that of DA (Fig 3) This common binding mode for ligands with a catechol moiety was characterized by a tight bidentate bonding of the catechol oxygen atoms to the catalytic iron The mean Fig TH inhibition by NMNorsal and structurally related TIQs (A) Activity of recombinant hTH in the presence of DA and TIQs Data are shown as the percentage of the activity level in the absence of inhibitors (n = 4) (B) A Lineweaver–Burk plot of hTH activity in the presence of NMNorsal (1.0 lM) at various concentrations of the pterin cofactor DPH4 (n = 3) FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS 2111 TIQs imitate mechanisms of TH feedback inhibition J Scholz et al Fig 6,7-Dihydroxylated TIQs and DA bind at identical sites in the catalytic center of TH The ball and stick view shows the energetically most favorable conformations of NMNorsal (red), NMSal (yellow) and Norsal (green) in the crystal structure of the enzyme’s catalytic domain (Protein Data Bank identification code 2toh) [18] as determined by molecular docking; superposed is the binding position of DA (dark blue) [25] In these bidentate conformations, the immediate environment surrounding the active site iron (amber) was a slightly distorted octahedral shape, formed by the two catechol oxygens of the TIQs, a water molecule, and the TH residues His331, His336 and Glu376 NMTIQ (light blue) and the exogenous neurotoxin MPTP (orange) bound at a greater distance from the catalytic iron The nitrogen atoms of TH residues are colored blue, oxygen atoms are shown in red; hydrogen atoms are omitted for clarity distance between the catechol oxygen atoms and the ˚ iron was 1.74 A for both NMNorsal and Norsal and ˚ ˚ 1.72 A for NMSal, compared to 1.71 A for DA (Table 1) The oxygen atoms were placed opposite the e nitrogen atoms of His331 and His336, creating a plane perpendicular to the benzene ring of Phe300 The two oxygen atoms thus formed an intrinsic part of the iron coordination sphere, with the piperidine rings of the TIQs and the aminoethyl moiety of DA projecting from the binding pocket (Fig 3) A potential inter2112 action between the DA nitrogen and the backbone oxygen of Leu294 was outweighed by loss of rotational entropy of the DA side chain Table summarizes the energy components that characterize the most favorable conformations of NMNorsal, NMSal, Norsal and DA Electrostatic (Coulomb) interactions with the active site iron and the surrounding TH amino acids were the largest energy contribution in all conformations Separate docking runs for the (R) and (S) enantiomers of protonated NMNorsal and NMSal, respectively, revealed no differences in their binding sites or conformational energy components The binding site of the 6,7-dihydroxylated TIQs and DA interfered with that of the essential pterin cofactor [18,25], preventing the cofactor from gaining access to the active site In contrast, the energetically favored positions of the non-catechol compounds NMTIQ and MPTP (Fig 3) indicated a placement corresponding to the binding site of the amino acid substrate in the crystal structure of phenylalanine hydroxylase [26] In these conformations, hydrogen bonds formed between the positively charged nitrogen atoms of NMTIQ and MPTP and the backbone oxygen of Ser324 The distances between the nitrogen atoms and the oxygen of ˚ ˚ Ser324 were 2.01 A for NMTIQ and 2.26 A for MPTP, respectively Substantially greater distances ˚ ˚ (5.49 A for NMTIQ and 4.92 A for MPTP) hindered an alternative formation of hydrogen bonds between the nitrogen atoms of the ligands and the backbone oxygen of Pro325 Although we did not directly compare the molecular interaction energies of NMTIQ and MPTP with those of the physiological substrate tyrosine, we hypothesize that the binding affinity of both ligands will be much lower because NMTIQ and MPTP lack the carboxylate group of the amino acid, which is likely to interact electrostatically with Arg316 Therefore, competition between NMTIQ or MPTP and tyrosine for the common binding site seems improbable 6,7-Dihydroxylated TIQs oxidize the catalytic iron The divalent state of the iron atom in the center of the catalytic site is an essential requirement for TH activity [24] Catecholamine inhibitors trap the iron in a ferric state, leading to inactivation of the enzyme [27] Using low-temperature electron paramagnetic resonance (EPR) spectroscopy, we examined the oxidation status and spin of the iron in hTH in the presence of DA and four structurally distinct TIQs The divalent iron of the unbound enzyme was EPRsilent Addition of DA produced a signal with g values of 7.1 and 4.8 (Fig 4A) This signal originated from FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS J Scholz et al TIQs imitate mechanisms of TH feedback inhibition Table Molecular interaction energies of DA, TIQs and MPTP in the crystal structure of TH (Protein Data Bank identification code 2toh) ND, not determined Ligand DA NMNorsal NMSal Norsal NMTIQ MPTP FlexX rank FlexX score Forcefield rank Forcefield score ˚ Number of comparable placements (rmsd < 1.4 A) Final LIECE score (kcalỈmol)1) Iron oxygen distance (mean) Van der Waals interactions (kcalỈmol)1) Coulomb interactions (kcalỈmol)1) Polar solvation contribution (kcalỈmol)1) Nonpolar solvation contribution (kcalỈmol)1) Intramolecular contribution (kcalỈmol)1) )16.21 498.81 29 )29.43 1.71 )4.29 338.05 309.58 )2.99 )0.69 21 )10.67 10 499.07 11 )27.39 1.74 )9.42 163.33 145.36 )2.46 )0.33 21 )10.57 498.16 )28.99 1.72 )10.53 237.04 218.51 )2.3 )0.43 23 )12.71 11 488.3 22 )25.88 1.74 )7.98 175.12 157.65 )2.6 )0.64 57 16 471.11 )2.96 ND )22.86 101.19 123.67 )2.44 )0.16 101 15 463.13 )3.83 ND )27.87 )58.7 85.06 )3.09 )0.62 the ground state Kramers’ doublet of a trivalent S = ⁄ spin system with a rhombicity parameter of E ⁄ D = 0.05, indicating that the iron had been oxidized to Fe(III) The same characteristic signal was detected in the presence of NMNorsal (Fig 4A), NMSal and Norsal We determined the proportion of oxidized iron after adding DA or these TIQs at a concentration equimolar to the hTH subunit concentration (220 ± 7.5 lm) Equimolar DA concentrations led to the generation of 80% high-spin Fe(III); NMNorsal produced 64% high-spin Fe(III), NMSal 78% and Norsal 76% (Fig 4B) Oxidation of the iron strongly indicates that 6,7-dihydroxylated TIQs, like DA, coordinate directly to the active site iron In contrast, adding an equimolar concentration of the non-hydroxylated NMTIQ caused only formation of nonspecific high-spin ferric iron, which accounted for less than 4% of the total hTH iron content (Fig 4B) TH reactivation by protein kinase A The primary mechanism of short-term TH regulation is post-translational modification of the catecholaminebound enzyme by protein kinases, which phosphorylate TH at serine residues of the N-terminal domain [22,23,28] Phosphorylation by protein kinase A (PKA) at Ser40, the most prominent of these regulatory sites, increases the dissociation rate of bound catecholamine inhibitors [29,30] Catecholamine removal facilitates Fe(III) reduction by tetrahydropterin, leading to an increase in Vmax of the enzyme reaction [31,32] We compared the effects of PKA on TH activity after inhibition with either NMNorsal or DA PKA did not change the basal enzyme activity when hTH was fully reconstituted with Fe(II) and concentrations of the pterin cofactor DPH4 were saturating (Fig 4C) However, tyrosine hydroxylation increased when PKA was added after hTH inhibition by 0.1 lm DA (Fig 4C) PKA likewise reactivated hTH after inhibition by NMNorsal Incubation of the enzyme with 0.1 lm NMNorsal reduced its activity to approximately 50% When PKA was added after the incubation, hTH activity was fully restored (Fig 4C) TIQs stabilize TH, albeit at a reduced level of activity DA and other catecholamines have a stabilizing effect on the conformation of TH [33] We therefore compared the thermostability of hTH at 37 °C in the presence of NMNorsal, Norsal and DA In the absence of these ligands, the specific activity of hTH decreased slowly but continuously After 20 min, the hTH activity was 69 ± 4% compared to baseline; after h, the remaining activity was reduced to 3% (Fig 5A) DA (20 lm) markedly accelerated the initial loss of hTH activity and decreased tyrosine hydroxylation to 23% within 10 In contrast to the uninhibited enzyme, the activity remained steady at this level for 90 (Fig 5A) Similar to DA, NMNorsal and Norsal (20 lm each) also provoked a fast initial decline of hTH activity but stabilized the activity at 59% and 48%, respectively, for h Even after 20 h at 37 °C, the enzyme activity was not completely lost, with residual activities of 28% and 23%, respectively, compared to 2% after 20 h of incubation with DA Surprisingly, a small transient increase in hTH activity occurred after h incubation in the presence of NMNorsal and Norsal (Fig 5A) DA is an unstable neurotransmitter (Fig 5B) Its autoxidation leads to the formation of DA quinone and is accompanied by the generation of hydrogen FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS 2113 TIQs imitate mechanisms of TH feedback inhibition J Scholz et al Fig Oxidation of the active site iron (A) Rapid freeze-quench EPR spectra of hTH in the presence of equimolar concentrations of DA or NMNorsal exhibited a characteristic signal at g values of 7.1 and 4.8 (arrowheads), indicating the formation of Fe(III) The signal at g = 4.3 stemmed from nonspecific high-spin ferric iron; a Cu(II) impurity caused the signal at g = The redox state of the iron remained unchanged after addition of NMTIQ (B) To determine the proportion of enzyme-bound iron converted to Fe(III), we compared the integrated absorption spectra with a mM Fe(III) cytochrome P450cam standard The assays contained 220 ± 7.5 lM hTH subunits fully reconstituted with Fe(II) and equimolar concentrations of NMNorsal, NMSal and Norsal Nonspecific high-spin ferric iron formed in the presence of the non-hydroxylated NMTIQ accounted for less than 4% of the total iron **P < 0.01 compared to DA or any of the other TIQs in a one-way ANOVA followed by Tukey’s test (C) Activity of hTH phosphorylated by PKA after inhibition with DA (0.1 lM) or NMNorsal (0.1 lM) *P < 0.05 for the difference between hTH activities in the absence and presence of PKA (unpaired t test) peroxide and reactive oxygen species (ROS) [34,35] Accumulation of DA quinone and ROS may contribute to the rapid initial loss of hTH activity that we observed during the incubation with DA [36] Hydrogen peroxide may also be generated by partial uncoupling of the pterin oxidation from the tyrosine hydroxylation [37] In the presence of iron, hydrogen peroxide is converted to a hydroxyl radical and hydroxide through the Fenton reaction [34,38] We examined the possible involvement of ROS in hTH inhibition by DA, NMNorsal and Norsal using catalase (EC 1.11.1.6), which converts hydrogen peroxide to oxygen and water Catalase (0.05 mgỈmL)1) slowed the initial DA-induced decrease in hTH activity without preventing the overall activity loss (Fig 5C) Cata- 2114 lase did not alter the hTH inhibition by NMNorsal or Norsal (Fig 5C), nor did it have an effect on the decline of hTH activity in the absence of inhibitors (data not shown) We conclude that hydrogen peroxide is formed and accelerates TH inhibition in the presence of DA; in contrast, hydrogen peroxide appears not to be involved in the TH inhibition by NMNorsal and Norsal, which are stable compounds compared to DA (Fig 5B) Discussion The catecholamines DA, norepinephrine and epinephrine regulate TH activity through two types of inhibition: reversible competition with the essential FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS J Scholz et al TIQs imitate mechanisms of TH feedback inhibition Fig Thermostability of hTH increases after DA and TIQ chelation (A) Recombinant hTH was incubated with 20 lM DA, NMNorsal or Norsal for 20 h at 37 °C Aliquots of the assay were removed at the indicated intervals to measure enzyme activity We carried out six independent measurements for the uninhibited enzyme and four for each inhibitor at every interval (B) Stability of DA, NMNorsal and Norsal during incubation at 37 °C in Hepes buffer containing 1.5 lM Fe(II) sulfate (n = 4) (C) Activity of hTH incubated with DA, NMNorsal or Norsal (20 lM each) in the presence of catalase (50 lgỈmL)1) Catalase slowed the initial loss of hTH activity caused by DA but had no effect on hTH inhibition by NMNorsal or Norsal (n = 4) tetrahydropterin cofactor and an almost irreversible blockade of TH activity by facilitating oxidation of the catalytic iron [21,23] As catecholamine-bound TH is thermally stable and resists proteolytic cleavage [33], the enzyme becomes trapped in an inactive state Using recombinant human TH, we show here that endogenous TIQs associated with PD mimic the mechanisms of catecholamine feedback inhibition: TIQs both compete with the tetrahydropterin for access to the active site and form a tight bidentate ligation to the iron atom in the center of the catalytic site, the latter prompting oxidation of the iron and consequent hTH inactivation TH inhibition by endogenous TIQs depends critically on 6,7-dihydroxylation of the benzene ring Only NMNorsal, NMSal and Norsal, which possess an intact catechol moiety, are inhibitors of hTH; hTH activity does not decrease in the presence of the non-hydroxylated NMTIQ NMNorsal was the strongest inhibitor among the 6,7-dihydroxylated TIQs studied Its IC50 of 0.3 lm nearly equals that of DA, suggesting that even small intracellular concentrations of NMNorsal are sufficient to produce a major effect on neurotransmitter synthesis In comparison, up to 104-fold higher concentrations of TIQs are required to cause cytotoxic blockade of the mitochondrial respiratory chain [4], indicating that 6,7-dihydroxylated TIQs primarily interfere with DA synthesis in PD rather than provoking neuronal degeneration Although the levels of 6,7-dihydroxylated TIQs in the substantia nigra and corpus striatum of patients with PD are unknown, a recent analysis indicated that the average concentration of NMSal in the substantia nigra, caudate nucleus and putamen of individuals without neurological or psychiatric disease is between 65 and 110 pmolỈg)1 [2] Salsolinol and NMNorsal are normally not detected in the CSF, but elevated levels of these TIQs of up to 60 pmolỈmL)1 were found in patients with PD [3,12], and the concentration of FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS 2115 TIQs imitate mechanisms of TH feedback inhibition J Scholz et al NMSal in the CSF of patients in PD is twice as high as in control individuals of a similar age [1] The increased CSF levels probably reflect a rise in the nigrostriatal concentration of 6,7-dihydroxylated TIQs that is sufficient to provoke TH inhibition Similar to the kinetics of TH inhibition by catecholamines [10,39], enzyme inhibition by NMNorsal is competitive with respect to the tetrahydropterin cofactor and noncompetitive with respect to tyrosine Previous molecular docking studies [25,40] and X-ray crystallography [18] indicate that BH4 and analogue pterins coordinate close to the active-site iron in TH, forming an aromatic p-stacking interaction with enzyme residue Phe300 In the modeled complex of the enzyme’s catalytic domain, NMNorsal ligation interfered with the docking site for BH4 By preventing the pterin cofactor from binding, NMNorsal blocks tyrosine hydroxylation at a critical reaction step [41] The natural pterin BH4 is considered to be the first substrate to bind at the TH active site, followed by molecular oxygen and tyrosine [42] Furthermore, electron transfer from the BH4 carbonyl oxygen to the molecular oxygen and the generation of a hydroxylating intermediate are presumably rate-limiting for the enzyme reaction [43,44] Direct binding of NMNorsal, NMSal and Norsal to the iron at the center of the catalytic site enabled bidentate ligation between the two hydroxyl residues of their catechol moiety and the iron The unbound ends of these molecules projected from the binding pocket Potentially, they interact with the enzyme’s regulatory domain, which has not been crystallized yet and was not incorporated in our model The coordination mode and actual binding site of the 6,7-dihydroxylated TIQs are identical with those for the catecholamine feedback inhibitor DA in a previous docking model [25] and an X-ray absorption fine-structure study [27] In our model, these TIQs had almost the same binding affinities to the catalytic center as DA In contrast, NMTIQ, which lacks a catechol moiety, occupied the binding site of the amino-acid substrate tyrosine [26] when docked into the catalytic center of TH The same conformation was obtained with MPTP However, neither NMTIQ nor MPTP formed electrostatic interactions with the surrounding TH residues, resulting in a low binding affinity NMTIQ is therefore unlikely to compete with tyrosine in vivo, which may explain why NMTIQ does not have an inhibitory effect on TH However, molecular docking in our model was limited to the catalytic center of TH, and the lack of a strong docking conformation here does not exclude the existence of allosteric binding sites for NMTIQ or MPTP 2116 Tight ligation of DA and other catecholamine end products to the active-site iron of TH has two major consequences First, catecholamine binding increases the proportion of oxidized iron bound to the enzyme [10,27,39,45], leading to loss of TH activity [46,47] Second, thermal stability of the enzyme increases and its resistance to proteolysis improves [33] TH preparations from animal tissues are inevitably contaminated with catecholamines and thus contain a sizable proportion of bound Fe(III) [45] In our study, we used recombinant hTH reconstituted with Fe(II), which allowed us to accurately quantify the formation of Fe(III) We found that equimolar concentrations of NMNorsal, NMSal or Norsal cause a rapid increase in Fe(III) In the presence of these TIQs, between 64 and 78% of the hTH iron was oxidized, compared to 80% in the presence of DA The precise mechanism responsible for the oxidation of enzyme-bound Fe(II) is unclear Most likely, molecular oxygen is the actual oxidant [48] Formation of a TH–Fe(III)–catecholamine complex induces an absorbance change at 700 nm that can be detected using visible spectroscopy Under anaerobic conditions, the absorbance change is only 50% of that observed in the presence of molecular oxygen [27] An important effect of catechols appears to be a shift in the equilibrium of bound iron towards the ferric state and prevention of its reduction to Fe(II) Consequently, catechols, which themselves are reducing agents, trap oxidized iron in the complex with the enzyme To study the effect of DA and 6,7-dihydroxylated TIQs on TH stability, we incubated the enzyme with DA, NMNorsal or Norsal at 37 °C for up to 20 h In the absence of inhibitors, hTH activity gradually declined DA rapidly reduced hTH activity by 77%, but the residual activity remained stable for 90 NMNorsal and Norsal also caused an initially rapid decrease in hTH activity; however, in the presence of these TIQs, residual activity levels of 59 and 48%, respectively, were sustained over several hours, exceeding the activity of the uninhibited enzyme DA is an unstable neurotransmitter and its metabolites are likely to contribute to the more profound loss of hTH activity during the incubation DA autoxidation produces DA quinone, which inhibits TH through covalent modification of its cysteinyl residues [36,49] Both autoxidation and enzymatic DA metabolism lead to the generation of hydrogen peroxide In the iron-rich environment of the substantia nigra, hydrogen peroxide is readily converted to ROS such as superoxide and hydroxyl radicals [34,38] Partial uncoupling of the hydroxylase reaction caused by ligands binding to the enzyme active site and changing its geometry may FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS J Scholz et al provide another source of ROS [37] Catalase protects against the formation of oxygen radicals by converting hydrogen peroxide to oxygen and water Catalase attenuated the loss of hTH activity during incubation with DA, but had no effect on hTH activity in the presence of NMNorsal or Norsal We conclude that hydrogen peroxide formation accelerates TH inhibition by DA, but is not involved in inhibition of the enzyme by 6,7-dihydroxylated TIQs The similar residual activity levels of the enzyme after 20 h of incubation with DA, NMNorsal or Norsal indicate that hydrogen peroxide is not required for the overall inhibitory effect of catechols, including DA Reactivation of catecholamine-bound TH is mediated by phosphorylation at serine residues within the N-terminal domain [22] Cyclic AMP-dependent phosphorylation at Ser40 by PKA does not directly regulate the reduction of Fe(III) [32], but strongly increases the dissociation rate of catecholamines and decreases the KM for the pterin cofactor [29,30,50,51] Phosphorylation at Ser40 also increases the affinity of TH for 14-3-3 proteins, which protect the enzyme from dephosphorylation [51] Catecholamine removal allows the pterin cofactor to regain access to the enzyme active site and reduce the iron to its active ferrous form [32,48] PKA also restored hTH activity after inhibition by NMNorsal Because NMNorsal and DA bind at identical sites in the catalytic center, we hypothesize that hTH phosphorylation by PKA results in a conformational change that facilitates the release of NMNorsal in the same way as it promotes the dissociation of catecholamine inhibitors However, the precise conformational changes that are induced by the phosphorylation of TH are unknown Phosphorylation at Ser40 may provide a negative charge that interacts with the amino group of DA and the pyridine moiety of NMNorsal in opposition to the bidentate ligation of the catalytic iron, pulling the ligands away from the iron and allowing them to leave the catalytic center; alternatively, phosphorylation-induced conformational changes may mimic protonation of an N-terminal TH residue that interacts with positively charged ligand groups, reducing their binding affinity [29,52] The increase in endogenous TIQs is most prominent in early PD [12,53], when approximately two-thirds of the dopaminergic neurons in the substantia nigra have been lost [54] Supported by other, non-dopaminergic compensatory mechanisms [55], the remaining neurons need to increase DA synthesis and release in order to balance the shortfall caused by their degenerating counterparts [14,15] We propose that blockade of catecholamine synthesis by NMNorsal and related endogenous TIQs enhances DA depletion Dopaminergic TIQs imitate mechanisms of TH feedback inhibition neurons of the substantia nigra are likely to be primarily affected, because endogenously formed 6,7-dihydroxylated TIQs accumulate in this region of the human midbrain [2] TH inhibition occurred at TIQ concentrations substantially lower than those required for blockade of mitochondrial respiration and induction of neuronal cell death [4–6] Even though the remarkable stabilizing effect of TIQs on purified TH may translate into preservation of a reduced enzyme activity in vivo, the predominant effect of NMNorsal and other 6,7dihydroxylated TIQs present in PD is likely to be a decrease in DA synthesis Experimental procedures Chemicals Chemical compounds were purchased from Sigma-Aldrich (Munich, Germany) unless otherwise indicated NMNorsal was synthesized by demethylation of 2-methyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline using 47% hydrogen bromide [56] The purity of the product was > 98% as determined by NMR spectroscopy Recombinant human TH isozymes Complementary DNAs of the coding sequences for hTH1 and hTH4 were inserted into a pET vector and transcribed in a BL21(DE3) strain of Escherichia coli engineered to contain an isopropyl b-d-thiogalactopyranoside (IPTG)-inducible T7 RNA polymerase gene [47] After incubation in the presence of 0.4 mm IPTG for h at 37 °C, the bacteria were harvested and stored at )20 °C until use We lysed the bacteria using a French press (Thermo Scientific, Waltham, MA, USA), and, after centrifugation at 35 000 g for h, purified the hTH isoforms from the supernatant as previously described [47], using a combination of diethylaminoethyl (DEAE)–Sepharose anion exchange chromatography, heparin–Sepharose affinity chromatography and size-exclusion chromatography on a Sephacryl S-300 gel column (GE Healthcare, Uppsala, Sweden) We verified by N-terminal amino acid sequence analysis that the isoforms were pure and had the predicted sequences [19] except for the N-terminal methionine residue, which was missing in 96% of hTH1 and 90% of hTH4 samples We concentrated the purified enzymes and stored them in liquid nitrogen The hTH isoforms typically contained less than 0.1 iron atoms per subunit, had a high catalytic activity when reconstituted with Fe(II), and were stable at neutral pH [47] TH activity assay We reconstituted recombinant hTH1 and hTH4 (subunit concentration 0.1 lm) using 0.1 mm Fe(II) sulfate before FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS 2117 TIQs imitate mechanisms of TH feedback inhibition J Scholz et al we preincubated hTH with DA or TIQs for 15 in 100 mm Hepes buffer (pH 7.0) containing mgỈmL)1 bovine catalase The enzyme reaction was started by addition of 0.1 mm l-tyrosine (Merck, Darmstadt, Germany), 0.1 mm 6,7-dimethyl-5,6,7,8-tetrahydropterin (DPH4), 0.1 mL)1 dihydropteridine reductase and 0.1 mm NADH After incubation for at 30 °C under aerobic conditions, we terminated the reaction by adding 1.1% perchloric acid For hTH activation by PKA, we reconstituted the protein samples with Fe(II) as described above and added 0.2 mgỈmL)1 bovine PKA, 0.4 mm MgCl2 and 0.1 mm ATP before starting the reaction [57] The concentration of the reaction product l-DOPA was measured by HPLC with electrochemical detection We used 2-methyl-3-(3,4-dihydroxyphenyl)-dl-alanine (50 nm) as an internal chromatography standard HPLC was performed at 30 °C using a C18 column (Eurospher RP18, particle size lm, column size 250 · 4.0 mm; Knauer, Berlin, Germany) and pre-column (35 · 4.0 mm; Knauer) The mobile phase consisted of a degassed solution containing 0.3 mm Na2-EDTA, 0.52 mm 1-Na-octane sulfate, 11.5% methanol and 0.1 m citrate buffer, pH 3.0 The detector cell operated at 0.8 V Nonenzymatic l-DOPA formation was determined using 0.1 mm d-tyrosine as substrate in the presence of the TH inhibitor a-methyl-l-para-tyrosine (0.1 mm) Enzymatic synthesis of l-DOPA was determined by subtracting the concentration of nonenzymatically formed l-DOPA from the total concentration [9,10] Molecular docking Ligand–protein complexes were based on the crystal structure of the enzyme’s catalytic domain (Protein Data Bank identification code 2toh) [18] after removal of co-crystallized 7,8-dihydrobiopterin and all water molecules except for HOH601, which completes the iron coordination sphere as a counterpart of Glu376 Residue 300 of TH was reverted to phenylalanine [58] We employed the software corina (version F; Molecular Networks, Erlangen, Germany) to generate 3D structures of the ligands, flexx (version 2.0.2; BioSolveIT, Sankt Augustin, Germany) for the ligand docking, and amber (version 8; Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA) to optimize complexes by force-field energy minimization [25] General amber force-field atom types and Gasteiger atomic charges were assigned to the ligand atoms [59] We limited the output to a maximum of 200 placements per ligand; the (R) and (S) enantiomers of NMNorsal and NMSal were treated separately The docking runs included all active site atoms within a ˚ radius of 12.0 A around the catalytic iron The maximum distance between the hydroxyl oxygen atoms of the ligands ˚ and the active site iron was set at 5.0 A, allowing docking modes that included monodentate and bidentate binding [25] Ligand–protein complexes were subjected to 50 steps 2118 of steepest-descent energy minimization, followed by 350 steps of conjugated-gradient energy minimization, applying a distance-dependent dielectric constant of r In order to focus on the most plausible placements, the resulting conformations were clustered based on their rmsd values and force-field energies Starting from the energetically most favorable conformation as the reference placement, all con˚ formations with an rmsd of less than 1.4 A with respect to the reference placement were considered identical and excluded from further analyses This continued with the next best conformation of the remaining placements until no further placements were left We ranked alternative ligand placements according to the energy score of each conformation, and determined interaction energies for the 20 energetically most favorable conformations To account for aqueous solvation effects, we assessed electrostatic interactions using the generalized Born method The linear interaction energy with continuum electrostatics (LIECE) [60] was calculated as the sum of unweighted differences of van der Waals energies, electrostatic energies, electrostatic and nonpolar solvation energies, and an entropically reasoned penalty of 1.4 kJỈmol)1 per rotatable bond in order to estimate the relative binding affinities For iron, the surface parameters for the generalized Born calculations were unavailable and had to be estimated Electron paramagnetic resonance spectroscopy We reconstituted recombinant hTH samples with Fe(II) and incubated them with equimolar concentrations of DA or TIQs for under aerobic conditions at room temperature We recorded rapid freeze-quench EPR spectra at a temperature of 10 K and a microwave frequency of 9.6456 GHz using a conventional X-Band spectrometer (Bruker 200D SRC, Karlsruhe, Germany) equipped with a helium-flow cryostat (ESR 910, Oxford Instruments, Witney, UK) [27] The microwave power was 80 mW The modulation amplitude was 0.5 mT and the modulation frequency was 100 kHz Spin quantifications were performed by integration of the experimental absorption spectra and comparison with a mm Fe(III) cytochrome P450cam (camphor 5-monooxygenase) standard from Pseudomonas putida The integrated areas were weighted using Aasa correction factors [27] TH thermostability Recombinant hTH (2 lm) was incubated with 20 lm DA, NMNorsal or Norsal at 37 °C in the presence of 1.5 lm Fe(II) sulfate Catalase was included in the assay as indicated At defined intervals, we removed aliquots of lL to measure TH activity We incubated the aliquots for at 30 °C in a reaction mixture containing 50 mm Hepes buffer (pH 7), 0.1 mm Fe(II) sulfate, 25 lm 3H-tyrosine and 50 lgỈmL)1 catalase The enzyme reaction was started by the addition of 0.5 mm BH4 in mm dithiothreitol, and FEBS Journal 275 (2008) 2109–2121 ª 2008 The Authors Journal compilation ª 2008 FEBS J Scholz et al stopped using 7.5% charcoal in m hydrochloric acid Using HPLC, we also determined the stability at 37 °C of DA, NMNorsal and Norsal during incubation for up to 20 h in Hepes buffer containing 1.5 lm Fe(II) sulfate Statistical analysis Data are given as mean ± SEM Mean differences between hTH activities in the absence and presence of PKA were analyzed by an unpaired t test We used a one-way anova followed by Tukey’s test to compare the proportions of hTH iron oxidized in the presence of DA and the indicated TIQs Acknowledgements We thank Englbert Bauml (Institute of Chemistry, ă University of Lubeck, Germany) for providing us with ă NMNorsal and Katharina Schnackenberg (Neurochemistry Research Group, Department of Neurology, University of Lubeck, Germany) for technical assisă tance The project was supported by the Medical Faculty of the University of Lubeck, Germany ă (MUL J031) and the Research Council of Norway References Maruyama W, Abe T, Tohgi H, Dostert P & Naoi M (1996) A dopaminergic neurotoxin, (R)-N-methylsalsolinol, increases in Parkinsonian cerebrospinal fluid Ann Neurol 40, 119–122 Maruyama W, Sobue G, Matsubara K, Hashizume Y, Dostert P & Naoi M (1997) A dopaminergic 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TH inhibition Similar to the kinetics of TH inhibition by catecholamines [10,39], enzyme inhibition by NMNorsal is competitive with respect to the tetrahydropterin cofactor and noncompetitive with. .. conformation of TH [33] We therefore compared the thermostability of hTH at 37 °C in the presence of NMNorsal, Norsal and DA In the absence of these ligands, the specific activity of hTH decreased slowly... plete loss of DA in the absence of neuronal degeneration [6] The CSF levels of TIQs increase in early PD and decrease as the disease progresses [12] TH inhibition by endogenous TIQs may therefore