Modulation by basic polypeptides of ATP-induced activation of tyrosine hydroxylase prepared from bovine adrenal medulla

The effects of basic polypeptides on the activation of adrenal tyrosine hydroxylase by ATP were investigated to show a possible involvement of macromolecular cell components in the regulation of the enzyme activity. Basic polypeptides caused an enhancement of the activation of tyrosine hydroxylase by low concentrations of ATP, and the potentiating effects of these polypeptides were observed to be dependent on their concentrations. Kinetic studies showed that basic polypeptides caused an increase in the Vmax of the ATP-activated enzyme for the cofactor without any change in the Km. These results suggest that basic polypeptides convert the enzyme from a nonsusceptible form to a form susceptible to ATP, thus resulting in the potentiation of the ATP-induced activation. Furthermore, the activation by ATP of tyrosine hydroxylase was not observed after treatment of the enzyme preparation with CM-cellulose, and the responsiveness of the enzyme treated with CM-cellulose to ATP was partially restored by addition of basic polypeptides. These observations suggest the possibility that macromolecular cell components, presumably basic proteins, may be involved in the regulation of the activity of tyrosine hydroxylase through their modulating effects on the sensitivity of the enzyme to ATP within the cell.     

Mechanism of tyrosine hydroxylase activation by phosphorylation

It was found that the fluorescence of 1,N⁶-ethenoadenosine triphosphate (ε-ATP) bound to myosin subfragment-1 (S-1) is resistant to quenching by acrylamide, while free ε-ATP is effectively quenched. Thus in the presence of acrylamide the bound ε-ATP is still highly fluorescent, while free ε-ATP is much less fluorescent. The Stern-Volmer constants of bound and free ε-ATP are 6.83 and 57.86 M^?1, respectively. Therefore it is easy to distinguish spectro-scopically the nucleotide-ligated S-1 from nucleotide-free S-1. Moreover acrylamide does not alter the S-1-Mg²⁺-ε-ATPase behavior.     

Inactivation of tyrosine hydroxylase by reduced pterins

Tyrosine hydroxylase [E.C. 1.14.16.2] is inactivated by incubation with its reduced pterin cofactors L-erythro-tetrahydrobiopterin, 2-amino-4-hydroxy-6-methyl-5,6,7,8-tetrahydropterin and 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropterin. Each of the two diastereoisomers of L-erythro-tetrahydrobiopterin inactivates tyrosine hydroxylase but the natural (6R) form is much more potent than the unnatural (6S) form at equimolar concentrations. The pterin analog 6-methyl-5-deazatetrahydropterin, which has no cofactor activity, also inactivates the enzyme whereas the oxidized pterins 7,8 dihydrobiopterin and biopterin do not. The inactivation process is both temperature and time dependent and results in a reduction of the Vmax for both tetrahydrobiopterin and tyrosine. Neither tyrosine nor oxygen inactivates tyrosine hydroxylase.     

Mechanism of oxygen activation by tyrosine hydroxylase

The mechanism by which the tetrahydropterin-requiring enzyme tyrosine hydroxylase (TH) activates dioxygen for substrate hydroxylation was explored. TH contains one ferrous iron per subunit and catalyzes the conversion of its tetrahydropterin cofactor to a 4a-carbinolamine concomitant with substrate hydroxylation. These results are in accord with shared mechanisms of oxygen activation by TH and the more commonly studied tetrahydropterin-dependent enzyme phenylalanine hydroxylase (PAH) and strongly suggest that a peroxytetrahydropterin is the hydroxylating species generated during TH turnover. In addition, TH can also utilize H2O2 as a cofactor for substrate hydroxylation, a result not previously established for PAH. A detailed mechanism for the reaction is proposed. While the overall pattern of tetrahydropterin-dependent oxygen activation by TH and PAH is similar, the H2O2-dependent hydroxylation performed by TH provides an indication that subtle differences in the Fe ligand field exist between the two enzymes. The mechanistic ramifications of these results are briefly discussed.     

The pH dependence of binding of inhibitors to bovine adrenal tyrosine hydroxylase

The inhibition of purified bovine adrenal tyrosine hydroxylase by several product and substrate analogues has been studied to probe the kinetic mechanism. Norepinephrine, dopamine, and methylcatechol are competitive inhibitors versus tetrahydropterins and noncompetitive inhibitors versus tyrosine. 3-Iodotyrosine is an uncompetitive inhibitor versus tetrahydropterins and a competitive inhibitor versus tyrosine. The Ki value for 3-iodotyrosine depends on the tetrahydropterin used. These results are consistent with tetrahydropterin binding first to the free enzyme followed by binding of tyrosine. 5-Deaza-6-methyltetrahydropterin is a noncompetitive inhibitor versus tetrahydropterins and tyrosine. The effect of varying the concentration of tyrosine on the Ki value for 5-deaza-6-methyltetrahydropterin is consistent with the binding of this inhibitor to both the free enzyme and to an enzyme-dihydroxyphenylalanine complex. Dihydroxyphenylalanine also is a noncompetitive inhibitor versus tetrahydropterins and tyrosine; the effect of changing the fixed substrate is consistent with the binding of this inhibitor to both the free enzyme and to the enzyme-tetrahydropterin complex. The effect of pH on the Ki values was determined in order to measure the pKa values of amino acid residues involved in substrate binding. Tight binding of catechols requires that a group with a pKa value of 7.6 be deprotonated. Binding of 3-iodotyrosine involves two groups with pKa values of 7.5 and about 5.5, one of which must be protonated for binding. Binding of 5-deaza-6-methyltetrahydropterin requires that a group on the free enzyme with a pKa value of 6.1 be protonated. The Ki value for dihydroxyphenylalanine is relatively insensitive to pH, but the inhibition pattern changes from noncompetitive to competitive above pH 7.5, consistent with the measured pKa values for binding to the free enzyme and to the enzyme-tetrahydropterin complex.     

Inhibition of phenylalanine hydroxylase activity by alpha-methyl tyrosine, a potent inhibitor of tyrosine hydroxylase.

Inhibitors of phenylalanine hydroxylase and tyrosine hydroxylase were used in the assay of phenylalanine hydroxylase in liver and kidney of rats and mice. Parachlorophenylalanine (PCPA), methyl tyrosine methyl ester and dimethyl tyrosine methyl ester showed 5–15% inhibition while α-methyl tyrosine seemed to inhibit phenylalanine hydroxylase to the extent of 95–98% at concentrations of 5 × 10 ⁻⁵M –1 × 10 ⁻⁴M. After a phenylketonuric diet (0.12% PCPA + 3% excess phenylalanine), the liver showed 60% phenylalanine hydroxylase activity and kidney 82% that present in pair-fed normals. Hepatic activity was normal after 8 days refeeding normal diet whereas kidney showed 63% of normal activity. The PCPA-fed animals showed 34% in liver and 38% in kidney as compared to normals; in both cases normal activity was noticed after refeeding. The phenylalanine-fed animals showed activity similar to that seen in phenylketonuric animals. The temporary inducement of phenylketonuria in these animals may be due to a slight change in conformation of the phenylalanine hydroxylase molecule; once the normal diet is resumed, the enzyme reverts back to its active form. This paper also suggests that α-methyl tyrosine when fed in conjunction with the phenylketonuric diet may suppress phenylalanine hydroxylase activity completely in the experimental animals thus yielding normal tyrosine levels as seen in human phenylketonurics.     

Nitration and inactivation of tyrosine hydroxylase by peroxynitrite

Tyrosine hydroxylase (TH) is modified by nitration after exposure of mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydrophenylpyridine. The temporal association of tyrosine nitration with inactivation of TH activity in vitro suggests that this covalent post-translational modification is responsible for the in vivo loss of TH function (Ara, J., Przedborski, S., Naini, A. B., Jackson-Lewis, V., Trifiletti, R. R., Horwitz, J., and Ischiropoulos, H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7659-7663). Recent data showed that cysteine oxidation rather than tyrosine nitration is responsible for TH inactivation after peroxynitrite exposure in vitro (Kuhn, D. M., Aretha, C. W., and Geddes, T. J. (1999) J. Neurosci. 19, 10289-10294). However, re-examination of the reaction of peroxynitrite with purified TH failed to produce cysteine oxidation but resulted in a concentration-dependent increase in tyrosine nitration and inactivation. Cysteine oxidation is only observed after partial unfolding of the protein. Tyrosine residue 423 and to lesser extent tyrosine residues 428 and 432 are modified by nitration. Mutation of Tyr(423) to Phe resulted in decreased nitration as compared with wild type protein without loss of activity. Stopped-flow experiments reveal a second order rate constant of (3.8 +/- 0.9) x 10(3) m(-1) s(-1) at pH 7.4 and 25 degrees C for the reaction of peroxynitrite with TH. Collectively, the data indicate that peroxynitrite reacts with the metal center of the protein and results primarily in the nitration of tyrosine residue 423, which is responsible for the inactivation of TH.     

Modification of the tyrosine hydroxylase assay

A modification of the tyrosine hydroxylase assay is described in which ascorbate, rather than 2-mercaptoethanol or dihydropteridine reductase with NADPH, is used as the reductant. Enzyme activity is 3–4 times higher with ascorbate than with the other reducing agents. Low blanks are obtained with the ascorbate system provided that catalase is also included. The tissue distribution and kinetic activation of the enzyme have been studied with the ascorbate assay. The results obtained are consistent with the biological and regulatory properties of the enzyme which have been determined with the other reducing systems.     

Phosphorylation of tyrosine hydroxylase by calmodulin-dependent multiprotein kinase

Tyrosine hydroxylase purified from rat pheochromocytoma was phosphorylated stoichiometrically by either cyclic AMP-dependent protein kinase or calmodulin-dependent multiprotein kinase from skeletal muscle, but not by five other protein kinases tested. The activity of tyrosine hydroxylase was elevated 3-fold by cyclic AMP-dependent protein kinase, but no activation was observed after phosphorylation by calmodulin-dependent multiprotein kinase. Phosphorylation produced by cyclic AMP-dependent protein kinase and calmodulin-dependent multiprotein kinase was additive, suggesting different sites of phosphorylation. This was confirmed by high-performance liquid chromatography analysis of tryptic phosphopeptides which demonstrated that the major sites phosphorylated by each protein kinase were distinct. A calmodulin-dependent multiprotein kinase that had identical properties and substrate specificity to the skeletal muscle enzyme was partially purified from rat pheochromocytoma. The possibility that this protein kinase is involved in the regulation of tyrosine hydroxylase activity in adrenergic tissue in vivo is discussed.     

Modulation of monocytic differentiation of HL-60 cells by inhibitors of protein tyrosine kinases.

We showed previously that the expressions of various src family protein tyrosine kinases (PTKs) were induced independently during the monocytic differentiation of HL-60 cells. The role of PTKs was further assessed in the present study by investigating the effects of PTK inhibitors on the differentiation. It was demonstrated that PTK inhibitors such as genistein and herbimycin A modulated monocytic differentiation of HL-60 cells; they inhibited the differentiation induced by TPA, while promoting that induced by vitamin D3 (D3). Immunoblotting analysis of protein molecules which had been phosphorylated on their tyrosine residues demonstrated that TPA induced phosphorylation of certain molecules different from those induced by D3 in HL-60 cells. PTK inhibitors blocked the phosphorylation and modulated differentiation driven by the inducers. These data suggest that PTKs are involved both promotively and suppressively in signaling events that induce monocytic differentiation of HL-60 cells.     

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.     

Purification of tyrosine hydroxylase by high-pressure liquid chromatography

Our study of tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) from rabbit adrenals has identified two major requirements which are likely to be of general application for the optimal purification and recovery of enzyme activity consequent to high-pressure liquid chromatography: (i) recovery of activity is maximized by pretreatment of the high-pressure liquid chromatography column before each use with protein to saturate high affinity, nonspecific sites exposed by the methanol used for washing, and storage of the column. (ii) Both purification and recovery are critically dependent upon the molarity of the mobile phase buffer. Examination of high-pressure liquid chromatography purified rabbit adrenal tyrosine hydroxylase by nondenaturing gel electrophoresis indicated that tyrosine hydroxylase activity was associated with one of the two protein bands in the gel. Thus, the convenient purification procedure described in this report leads to preparative amounts of tyrosine hydroxylase which is approximately 50% homogeneous.     

Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.     

Antioxidative galloyl esters as enzyme inhibitors of p-hydroxybenzoate hydroxylase

Gallic acid and its esters were evaluated as enzyme inhibitors of recombinant p-hydroxybenzoate hydroxylase (PHBH), a NADPH-dependent flavin monooxygenase from Pseudomonas aeruginosa. n-Dodecyl gallate (DG) (IC(50)=16 microM) and (-)-epigallocatechin-3-O-gallate (EGCG) (IC(50)=16 microM), a major component of green tea polyphenols, showed the most potent inhibition, while product-like gallic acid did not inhibit the enzyme significantly (IC(50)>250 microM). Inhibition kinetics revealed that both DG and EGCG inhibited PHBH in a non-competitive manner (K(I)=18.1 and 14.0 microM, respectively). The enzyme inhibition was caused by specific binding of the antioxidative gallate to the enzyme, and by scavenging reactive oxygen species required for the monooxygenase reaction. Molecular modeling predicted that EGCG binds to the enzyme in the proximity of the FAD binding site via formation of three hydrogen bonds.