Recombinant Human Tyrosine Hydroxylase Research Articles

Serine 19 phosphorylation and 14-3-3 binding regulate phosphorylation and dephosphorylation of tyrosine hydroxylase on serine 31 and serine 40.

Multisite phosphorylation and structural flexibility allow for complex regulation of proteins through cellular signaling. Tyrosine hydroxylase (TH), a key enzyme of catecholamine synthesis, is regulated by multiple neuronal signaling pathways through phosphorylation at serine 19 (Ser19), serine 31 (Ser31), and serine 40 (Ser40) located in the flexible, far N-terminal region of the regulatory domain. Phosphorylated Ser19 (pSer19) provides a binding site for 14-3-3 proteins, a family of multi-target binding adaptor proteins. We hypothesized that pSer19 and 14-3-3 binding can regulate access to the Ser31 and Ser40 sites and modulate the dynamics of their phosphorylation state. To avoid complications from upstream signal interactions and have good control of TH-phosphorylation and 14-3-3 binding stoichiometry, we used purified recombinant human TH and 14-3-3 dimer types. We found that pSer19 strongly stimulated Ser31 phosphorylation (4.6-fold), but inhibited pSer31 dephosphorylation (3.4-fold). Binding of 14-3-3ζ counteracted the stimulatory effect of pSer19 on phosphorylation at Ser31, but amplified the effect on its dephosphorylation. In contrast, phosphorylation at Ser19 had moderate effect on pSer40 dephosphorylation, but 14-3-3ζ binding inhibited dephosphorylation, an effect that was consistent across different homo- and heterodimeric 14-3-3s. Additional phosphorylation of Ser31 or Ser40 had little impact on the binding affinity of pSer19 TH to 14-3-3s. Mathematical modeling was performed to elucidate possible physiological implications of these observations. We propose a role of Ser19 and 14-3-3 proteins as modulators of TH phosphorylation in response to neuronal co-signaling events. These mechanisms add to our understanding of the multifaceted roles of phosphorylation and adaptor proteins in cellular signaling.

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson’s disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ∼23mg of pure, active and stable enzyme exhibiting a specific activity of ∼17nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson’s disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.

Molecular Mechanism for Pterin-Mediated Inactivation of Tyrosine Hydroxylase: Formation of Insoluble Aggregates of Tyrosine Hydroxylase

Tyrosine hydroxylase (TH), an iron-containing enzyme, catalyzes the first and rate-limiting step of catecholamine biosynthesis, and requires tetrahydrobiopterin (BH4) as a cofactor. We found that preincubation of recombinant human TH with BH4 results in the irreversible inactivation of the enzyme at a concentration far less than the Km value toward BH4 in spite of its cofactor role, whereas oxidized biopterin, which has no cofactor activity, does not affect the enzyme activity. We show that TH is inactivated by BH4 in competition with the binding of dopamine. The sequential addition of BH4 to TH results in a gradual decrease in the intensity of the fluorescence and CD spectra without changing their overall profiles. Sedimentation velocity analysis demonstrated an association of TH molecules with each other in the presence of BH4, and studies using gel-permeation chromatography, turbidity measurements, and transmission electron microscopy demonstrated the formation of amorphous aggregates with large molecular weights following the association of the TH proteins. These results suggest that BH4 not only acts as a cofactor, but also accelerates the aggregation of TH. We propose a novel mechanism for regulating the amount of TH protein, and discuss its physiological significance.

Regulation of tyrosine hydroxylase by stress-activated protein kinases.

Recombinant human tyrosine hydroxylase (hTH1) was found to be phosphorylated by mitogen and stress-activated protein kinase 1 (MSK1) at Ser40 and by p38 regulated/activated kinase (PRAK) on Ser19. Phosphorylation by MSK1 induced an increase in Vmax and a decrease in Km for 6-(R)-5,6,7,8-tetrahydrobiopterin (BH4), while these kinetic parameters were unaffected as a result of phosphorylation by PRAK. Phosphorylation of both Ser40 and Ser19 induced a high-affinity binding of 14-3-3 proteins, but only the interaction of 14-3-3 with Ser19 increased the hTH1 activity. The 14-3-3 proteins also inhibited the rate of dephosphorylation of Ser19 and Ser40 by 82 and 36%, respectively. The phosphorylation of hTH1 on Ser19 caused a threefold increase in the rate of phosphorylation of Ser40. These studies provide new insights into the possible roles of stress-activated protein kinases in the regulation of catecholamine biosynthesis.

Modification of tyrosine hydroxylase activity by chloral derived beta-carbolines in vitro.

Beta-carbolines have been suggested to be involved in the pathogenesis of Parkinson's disease as a result of their structural similarity to the neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The chloral-derived beta-carboline derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) causes cell loss in neuronal and glial cell cultures and induces a slowly developing neurodegenerative process in rats. In our experiments, effects of TaClo and its derivatives 2-methyl-TaClo (2-Me-TaClo), and 1-dichloromethylene-1,2,3,4-tetrahydro-beta-carboline (1-CCl(2) -THbetaC) on tyrosine hydroxylase (TH) activity were investigated in TH assays using homogenate preparations of the rat nucleus accumbens and recombinant human TH (hTH1). TH activity was determined in vitro by measuring l-DOPA production with HPLC-ECD. Using homogenate preparations, TaClo, 2-Me-TaClo, and 1-CCl(2) -THbetaC inhibited TH in concentrations of 0.1 mm, while 1-CCl(2) -THbetaC in low concentrations enhanced TH activity. When TH was activated by PACAP-27, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC also inhibited activated enzyme activity in high concentrations. However, in the case of 2-Me-TaClo and 1-CCl(2) -THbetaC a biphasic effect was observed with a marked increase of TH activity in the nanomolar range. In our experiments using recombinant hTH1, TaClo, 2-Me-TaClo, or 1-CCl(2) -THbetaC did not modify enzyme activity. After activation of hTH1 by PKA all the tetrahydro-beta-carbolines investigated in this study decreased l-DOPA formation. We suggest that these beta-carbolines modulate dopamine synthesis by interacting with a protein kinase TH-activating system.

The binding of tyrosine hydroxylase to negatively charged lipid bilayers involves the N-terminal region of the enzyme

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of catecholamines. We have studied the association of recombinant human TH with model membranes by using either liposomes or silica gel beads coated with single phospholipid bilayers (TRANSIL ®). The use of TRANSIL beads has allowed the determination of apparent dissociation constants ( K d) for the binding of the enzyme to negatively charged bilayers ( K d=230–380 μM, at pH 6.0–7.0). Binding to the bilayers is accompanied by a decrease in enzyme activity. Proteolysed forms of the enzyme show decreased binding affinity and two putative amphipathic N-terminal α-helices are proposed to be involved in membrane binding. As seen by circular dichroism, binding to the bilayer does not seem to induce significant changes on the secondary structure content of the enzyme, but α-helical structures appear to be stabilized against thermal denaturation in the membrane-bound state. Thus, amphitropism, a mechanism that regulates the function of peripheral proteins by weak binding to membrane lipids, may add to the factors that regulate both the activity and the stability of TH.

Ubiquitination of soluble and membrane-bound tyrosine hydroxylase and degradation of the soluble form.

Tyrosine hydroxylase (TH) demonstrates by two-dimensional electrophoresis a microheterogeneity both as a soluble recombinant human TH (hTH1) and as a membrane-bound bovine TH (bTHmem). Part of the heterogeneity is likely due to deamidation of labile asparagine residues. Wild-type (wt)-hTH1 was found to be a substrate for the ubiquitin (Ub) conjugating enzyme system in a reconstituted in vitro system. When wt-hTH1 was expressed in a coupled transcription-translation TnT(R)-T7 reticulolysate system 35S-labelled polypeptides of the expected molecular mass of native enzyme as well as both higher and lower molecular mass forms were observed. The amount of high-molecular-mass forms increased by time and was enhanced in the presence of Ub and clasto-lactacystin beta-lactone. In pulse-chase experiments the amount of full-length hTH1 decreased by first-order kinetics with a half-time of 7.4 h and 2.1 h in the absence and presence of an ATP-regenerating system, respectively. The ATP-dependent degradation was inhibited by clasto-lactacystin beta-lactone. Our findings support the conclusion that hTH1 is ubiquitinated and at least partially degraded by the proteasomes in the reticulocyte lysate system. Finally, it is shown that the integral TH of the bovine adrenal chromaffin granule membrane (bTHmem) is ubiquitinated, most likely monoubiquitinated. Additional Ub-conjugates of this membrane, detected by Western blot analysis, have not yet been identified.

A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase.

Tetrahydropterins are obligatory cofactors for tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. A series of synthetic analogues of 6(R)-L-erythro-5,6,7, 8-tetrahydrobiopterin (BH(4)) with different substituents in positions C2, N3, C4, N5, C6, C7, and N8 on the ring were used as active site probes for recombinant human TH. The enzyme tolerates rather bulky substituents at C6, as seen by the catalytic efficiency (V(max)/K(m)) and the coupling efficiency (mol of L-DOPA produced/mol of tetrahydropterin oxidized) of the cofactors. Substitutions at C2, C4, N5, and N8 abolish the cofactor activity of the pterin analogues. Molecular docking of BH(4) into the crystal structure of the catalytic domain of ligand-free rat TH results in complexes in which the pteridine ring pi-stacks with Phe300 and the N3 and the amino group at C2 hydrogen bonds with Glu332. The pteridine ring also establishes interactions with Leu294 and Gln310. The distance between C4a in the pteridines and the active site iron was 4.2 +/- 0.5 A for the ensemble of docked conformers. Docking of BH(4) analogues into TH also shows that the most bulky substituents at C6 can be well-accommodated within the large hydrophobic pocket surrounded by Ala297, Ser368, Tyr371, and Trp372, without altering the positioning of the ring. The pterin ring of 7-BH(4) shows proper stacking with Phe300, but the distance between the C4a and the active site iron is 0.6 A longer than for bound BH(4), a finding that may be related to the high degree of uncoupling observed for 7-BH(4).

Proton NMR Studies on the Conformation of the Pterin Cofactor Bound at the Active Site of Recombinant Human Tyrosine Hydroxylase

SummaryThe binding of L-erythro-7,S-dihydrobiopterin (BH2) in the presence of L-Phe to recombinant human tyrosine hydroxylase was studied by 1H-NMR spectroscopy. The distances (± 1.3 A) from the active site metal were estimated to be 5 .3-6.4 A for observable protons of BH2 and 7 .0-7.9 A for L-Phe protons. Due to the lack of constraints from the pyrimidine ring in BH2 , two families of conformers were computed, with an average distance from the C4a in the pterin ring to the iron of 3.7 A (family A) and S.S A (family B). The conformers for 6-methyl-tetrahydropterin bound to the enzyme at near anaerobic conditions in the absence of L-Phe were also grouped into two families, with C4a-metal distances of 3.0-4.0 A (A) and 7.0-7.5 A (B). Based on the estimated conformation of the enzyme bound 5,6,7,S-tetrahydro- 3-methylpterin, family A of conformers was selected for both oxidized and reduced pterins. Our results indicate that the pterin does not coordinate to the iron, which seems to be involved both in the binding and activation of O2 and in the C-O bond formation in the aromatic ring of the substrate.

Resonance Raman Studies of Catecholate and Phenolate Complexes of Recombinant Human Tyrosine Hydroxylase

Human tyrosine hydroxylase isoform 1 (hTH1) was expressed in Escherichia coli, purified as the apoenzyme, and reconstituted with iron. The resonance Raman spectra of hTH1 complexed with dopamine, noradrenaline, tyramine, and catechol have been studied and compared to those obtained for TH isolated from bovine adrenal glands or rat phaeochromocytoma tissue. A TH-phenolate complex is reported for the first time. Using dopamine selectively 18O-labeled in the 3-position or both 3- and 4-hydroxy positions, we have been able to assign unambiguously the origin of the low-frequency vibration bands: the band at 631 cm-1 involves the oxygen in the 4-position; the band at 592 cm-1 involves the oxygen in the 3-position, and the band around 528 cm-1 is shifted by both, suggesting a chelated mode vibration. A small shift of the 1275 cm-1 band and no shift of the 1320 cm-1 band were observed, showing that those two bands involve essentially ring vibrations of the catecholate moiety, rather than the C--O stretching vibration as previously suggested. The spectrum of the catechol-d6-hTH1 complex confirms this assignment. The resonance Raman spectra of the 54Fe, 56Fe, or 57Fe isotope-containing enzymes complexed with dopamine are virtually identical, showing that the component of the iron in the approximately 600 cm-1 vibrations is too small to be observed. These results provide a better understanding of the Raman properties of iron-catecholate complexes in this enzyme, as well as in other metalloproteins and model compounds.

Evidence for a functionally important histidine residue in human tyrosine hydroxylase

Recombinant human tyrosine hydroxylase isozyme 1 (hTH1) shows a time- and concentration-dependent loss of catalytic activity when incubated with diethylpyrocarbonate (DEP) after reconstitution with Fe(II). The inactivation follows pseudo-first order kinetics with a second order rate constant of 300 M(-1) min(-1) at pH 6.8 and 20°C and is partially reversed by hydroxylamine. The difference absorption spectrum of the DEP-modified vs native enzyme shows a peak at 244 nm, characteristic of mono-N-carbethoxy-histidine. Up to five histidine residues are modified per enzyme subunit by a five-fold excess of the reagent, and two of them are protected from inactivation by the active site inhibitor dopamine. However, derivatization of only one residue appears to be responsible for the inactivation. Thus, no inactivation by DEP was found when the apoenzyme was preincubated with this reagent prior to its reconstitution with Fe(II), modifying four histidine residues.

Recombinant Human Tyrosine Hydroxylase Types 1–4 Show Regulatory Kinetic Properties for the Natural (6R)-Tetrahydrobiopterin Cofactor1

Human tyrosine hydroxylase (hTH) exists in four isoforms. The four recombinant hTH isoenzymes types 1-4 (hTH1-4) produced in and purified from Escherichia coli showed regulatory kinetic properties for the natural (6R)-L-erythro-tetrahydrobiopterin (RBPH4) as a cofactor. In contrast, the unnatural cofactor (6S)-L-erythro-tetrahydrobiopterin (SBPH4) and a synthetic cofactor (6RS)-methyl-tetrahydropterin (6MPH4) showed usual kinetic characteristics with each of hTH1-4. Substrate inhibition by tyrosine was observed for each of hTH1-4 with natural RBPH4. Two different Km values for pterin cofactor were observed at a high concentration (200 microM) of L-tyrosine only with natural RBPH4, in contrast to a single Km value for unnatural SBPH4 or synthetic 6MPH4. The present results suggest that in the presence of relatively high concentrations (approximately 100 microM) of tyrosine in vivo, RBPH4 cofactor may have a regulatory role for the activity of all four human isoenzymes in vivo. We also found that recombinant hTH1 and 3 were more unstable than hTH2 and 4, suggesting that the 4-amino-acid insertion in hTH2 and 4 may be responsible for the relative stability of hTH2 and 4 isoenzymes.

Conformation and interaction of phenylalanine with the divalent cation at the active site of human recombinant tyrosine hydroxylase as determined by proton NMR

Recombinant human tyrosine hydroxylase has been purified as a metal-free apoenzyme (apo-hTH1) which tightly binds one Fe2+, Co2+, or Zn2+ per subunit with activation only by Fe2+ and competitive inhibition by the other cations. L-tyrosine and L-phenylalanine are alternative substrates for this enzyme, giving similar Vmax values, although the KM value for phenylalanine is about 8-fold greater than for tyrosine. Apo-hTH1 enhances the paramagnetic effects of Co2+ on 1/T1 and 1/T2 values of the protons of enzyme-bound phenylalanine both in the presence and in the absence of the oxidized form of the cofactor L-erythro-7,8-dihydrobiopterin (BH2), which was used as an inactive analog of the natural cofactor (6R)-1-erythro-tetrahydrobiopterin (BH4). No effects of hTH1-Zn2+ on 1/T1 or 1/T2 are found. From paramagnetic effects of hTH1-Co2+ on 1/T1 of phenylalanine protons at 250 and 600 MHz, in the presence of BH2, a correlation time (tau c) of 1.8 +/- 0.1 ps was found. Using this tau c value, and assuming that only one proton of the pairs H3,H5, and H2,H6 is experiencing the total paramagnetic effect (asymmetric limiting case), distances from enzyme-bound Co2+ to phenylalanine (+/- 1.2 A) of 6.1 A (H3 or H5), 6.3 A (H2 or H6), 7.0 A (H4), 7.3 A (H alpha), > or = 7.4 A (H beta-pro-S), and > or = 7.6 A (H beta-pro-R) were calculated. The distances to H3 or H5 and to H2 or H6 are slightly increased to 6.8 and 7.0 A, respectively, if each proton of both degenerate pairs equally experiences the paramagnetic effect of Co2+ (symmetric limiting case). These distances place the aromatic ring of phenylalanine in the second coordination sphere of the metal, which would permit an Fe-bound oxy or peroxy species to approach molecular contact with C3/C4, suggesting a direct role of Fe2+ in the hydroxylation reaction. The same correlation time and similar distances were found in the absence of BH2 with H4 of phenylalanine slightly closer to the metal. In the ternary hTH1-Zn(2+).BH2.phenylalanine complex, eight interproton distances in the enzyme-bound phenylalanine were determined by NOESY spectra at 600 MHz at 35-, 50-, and 75-ms mixing times. The conformation of enzyme-bound phenylalanine, consistent with the six Co(2+)-proton distances and the eight interproton distances, is partially extended with torsional angles chi 1 = 97 degrees +/- 3 degrees and chi 2 = -78 degrees +/- 2 degrees.

The incorporation of divalent metal ions into recombinant human tyrosine hydroxylase apoenzymes studied by intrinsic fluorescence and 1H-NMR spectroscopy.

Three isoforms of human tyrosine hydroxylase were expressed in Escherichia coli and purified to homogeneity as the apoenzymes (metal-free). The apoenzymes exhibit typical tryptophan fluorescence emission spectra when excited at 250-300 nm. The emission maximum (342 nm) was not shifted by the addition of metal ions, but reconstitution of the apoenzymes with Fe(II) at pH 7-9 reduced the fluorescence intensity by about 35%, with an end point at 1.0 iron atom/enzyme subunit. The fluorescence intensity of purified bovine adrenal tyrosine hydroxylase, containing 0.78 mol tightly bound iron/mol subunit, was reduced by only 6% on addition of an excess amount of Fe(II). Other divalent metal ions [Zn(II), Co(II), Mn(II), Cu(II) and Ni(II)] also reduced the fluorescence intensity of the human enzyme by 12-30% when added in stoichiometric amounts. The binding of Co(II) at pH 7.2 was also found to affect its 1H-NMR spectrum and this effect was reversed by lowering the pH to 6.1. The quenching of the intrinsic fluorescence of the human isoenzymes by Fe(II) was reversed by the addition of metal chelators. However, the addition of stoichiometric amounts of catecholamines, which are potent feedback inhibitors of tyrosine hydroxylase, to the iron-reconstituted enzyme, prevented the release of iron by the metal chelators. Fluorescence quenching, nuclear magnetic relaxation measurements and EPR spectroscopy all indicate that the reconstitution of an active holoenzyme from the isolated apoenzyme, with stoichiometric amounts of Fe(II) at neutral pH, occurs without a measurable change in the redox state of the metal. However, on addition of dopamine or suprastoichiometric amounts of iron, the enzyme-bound iron is oxidized to a high-spin Fe(III) (S = 5/2) form in an environment of nearly axial symmetry, thus providing an explanation for the inhibitory action of the catecholamines.

Regulation of recombinant human tyrosine hydroxylase isozymes by catecholamine binding and phosphorylation. Structure/activity studies and mechanistic implications.

Three isozymes of human tyrosine hydroxylase (hTH1, hTH2 and hTH4) were expressed in Escherichia coli and purified to homogeneity. Natural catecholamines and related synthetic compounds were found to be potent inhibitors, competitive to the tetrahydrobiopterin cofactor, of all the isozymes. Combining visible spectroscopy and equilibrium-binding studies, it was found that catecholamines bind to hTH1 and hTH2 with a stoichiometry of about 1.0 mol/mol enzyme subunit, interacting with the catalytic iron at the active site. All the isozymes tested were excellent substrates for cAMP-dependent protein kinase (Km = 5 microM, Vmax = 9.5 mumol.min-1.mg kinase-1). The incorporation of about 1.0 mol phosphate/subunit at Ser40 decreased the affinity of dopamine binding by a factor of 10. Conversely, the addition of stoichiometric amounts of Fe(II) and dopamine to the apoenzymes reduced both the affinity and stoichiometry of phosphorylation by cAMP-dependent protein kinase by 2-3-fold. These data provide evidence for a mutual interaction between the presumed regulatory and catalytic domains of hTH, and show that activation of the enzyme by phosphorylation and inactivation by binding of catecholamines are related events, which probably represent important mechanisms for the regulation of the enzyme activity in vivo.

Recombinant human tyrosine hydroxylase isozymes

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.