Effects of stereochemical structures of tetrahydrobiopterin on tyrosine hydroxylase.

1. Four stereochemical isomers of tetrahydrobiopterin, i.e., 6-L-erythro-, 6-D-erythro-, 6-L-threo-, or 6-D-threo-1,2-dihydroxypropyltetrahydropterin, have been synthesized and used as cofactors for tyrosine hydroxylase (EC 1.14.18.-) purified from the soluble fraction of bovine adrenal medulla. The L-erythro- (the putative natural cofactor) and D-threo isomers showed a striking similarity in their cofactor activities for tyrosine hydroxylase; the remaining two isomeric tetrahydrobiopterins, D-erythro and L-threo isomers, also had very similar cofactor characteristics. 2. The Km values of the L-erythro and D-threo isomers as cofactor were found to be dependent on their concentrations. When their concentrations were below 100 muM, the Km values of the L-erythro and D-threo isomers were fairly low (about 20 muM). However, the Km values were markedly higher (about 150 muM) at concentrations above 100 muM. The same kinetic behavior was also observed with the tetrahydrobiopterin prepared from a natural source (bullfrog). In contrast, the Km value of the L-threo or D-erythro isomer was found to be independent of the concentration and remained constant throughout the concentration examined. 3. The Km values of tyrosine did not show much difference (from 20 muM to 30 muM) with respect to the structure of the four isomeric cofactors. At high concentrations tyrosine inhibited the enzymatic reaction with any one of the four tetrahydrobiopterin cofactors. 4. Oxygen at high concentrations was also inhibitory with any one of the four stereochemical isomers as cofactor. Approximate Km values for oxygen with the tetrahydrobiopterins as cofactor were 1-5%. 5. In contrast to the four isomers of tetrahydrobiopterin, when 6-methyltetrahydropterin or 6,7-dimethyltetrahydropterin was used as cofactor tyrosine or oxygen did no inhibit the enzymatic reaction at high concentrations, and the Km values toward the pterin cofactor, tyrosine, and oxygen were significantly higher than the Km values with the tetrahydrobiopterins as cofactor.     

Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography

Tyrosine hydroxylase in bovine adrenal medulla was activated up to fourfold by incubation with low concentrations (15 micrograms/ml) of ribonucleic acids. At higher RNA concentrations, enzyme activity was inhibited. This interaction with RNA was exploited with the use of poly(A)-Sepharose and DNA-cellulose to effect a rapid purification of stable tyrosine hydroxylase from rat brain and bovine adrenal medulla in high yield (up to 58%). With the purified rat brain enzyme, RNA acted as an uncompetitive inhibitor, a concentration of 15 micrograms/ml lowering the Vmax of tyrosine hydroxylase from 1050 to 569 nmol min-1 mg-1 and lowering the Km for tyrosine from 6.1 to 3.6 microM. With the natural cofactor, tetrahydrobiopterin (BH4), two Km values were obtained, indicating the presence of two forms of the enzyme. Both Km values were decreased only slightly by RNA. The purified brain and adrenal enzymes both contained about 0.07 mol of phosphate/63,000-Da subunit; in both cases, cyclic AMP-dependent protein kinase catalyzed the incorporation of an additional 0.8 mol of phosphate/subunit. The purified enzyme also contains ribonucleic acid, which comprises about 10% of the total mass and appears to be important for full activity.     

The Activity of Rat Pineal and Brain Tyrosine Hydroxylase During the Daily Cycle of Light and Darkness as Determined by the Modified 14CO2 Assay Method

A previous published assay method for tyrosine hydroxylase by the evolution of 14CO2 was modified to a two-step procedure to allow reliable measurement of large numbers of samples containing low tyrosine hydroxylase activity. The reliability of the method was examined in detail. Properties of rat brain and pineal tyrosine hydroxylase solubilized with 0.2% Triton X-100 were as follows. The apparent Km values of the brain enzyme for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-L-erythro-biopterin (BPH4) as cofactor and for BPH4 with 62 microM-L-tyrosine as substrate were approximately 25 microM and 85 microM, respectively. The Km's for L-tyrosine with 1 mM-(6-DL)-5,6,7,8-tetrahydro-6-methylpterin (6MPH4) as cofactor and for 6MPH4 with 210 microM-L-tyrosine as substrate were 68 microM and 270 microM, respectively. The marked substrate inhibition by high concentrations of L-tyrosine was observed only when BPH4 was used as cofactor. High concentrations of BPH4 inhibited the reaction slightly. The kinetic properties of tyrosine hydroxylase in the pineal extract were similar to those of the brain enzyme, except that a Lineweaver-Burk plot of reciprocal velocity versus the reciprocal concentration of BPH4 with 62 microM-L-tyrosine as substrate deviated downward at a BPH4 concentration of about 100 microM. Analyses of the plot indicated that the peculiar kinetic property may represent either the reaction occurring at two independent sites or with two forms (6L- and 6D-isomers) of the tetrahydrobiopterin cofactor, with apparent Km for BPH4 of 23 microM and 1025 microM, respectively, or the negatively cooperative ligand binding with a Hill coefficient of 0.72. Based on the results obtained as reported above the standard assay conditions of tyrosine hydroxylase in tissue extracts were established. Using the assay method and conditions, the absence of the daily rhythmicity of tyrosine hydroxylase in rat pineal glands and three discrete brain areas was demonstrated. The findings, especially on pineal tyrosine hydroxylase, are discussed in relation to the daily change of noradrenaline turnover.     

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.     

The tyrosine-dependent oxidation of tetrahydropterins by lysolecithin-activated rat liver phenylalanine hydroxylase

In the presence of tyrosine, phenylalanine hydroxylase, which has been activated with lysolecithin, catalyzes the oxidation of tetrahydrobiopterin at a rate 10-20% that of the parallel reaction with phenylalanine. Unlike the reaction with phenylalanine, there is no net concomitant hydroxylation of tyrosine, although the amino acid is still a necessary component. Tyrosine appears to form an abortive complex with the activated enzyme, the pterin cofactor and molecular oxygen. The Km for tetrahydrobiopterin is identical for the reactions with phenylalanine and tyrosine, whereas the Km for tyrosine is approximately 3 1/2 times greater than the Km for phenylalanine. The tyrosine-dependent oxidation of tetrahydrobiopterin proceeds at both pH 6.8 and 8.2 and shows a similar dependence on the pH as that of the physiological reaction. Tetrahydrobiopterin can be replaced by the artificial cofactor, 6-methyltetrahydropterin, in the tyrosine-dependent oxidation at both pH 6.8 and 8.2. As in the parallel reaction with phenylalanine, both the Km for the cofactor and the Km for the aromatic amino acid increase with this substitution.     

Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S -nitroso- N -acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6 S -isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6 R -isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.     

The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.

Pheochromocytoma tyrosine hydroxylase was reported to have unusual catalytic properties, which might be unique to the tumor enzyme (Dix, T. A., Kuhn, D. M., and Benkovic, S. J. (1987) Biochemistry 24, 3354-3361). Two such properties, namely the apparent inability to hydroxylate phenylalanine and an unprecedented reactivity with hydrogen peroxide were investigated further in the present study. Tyrosine hydroxylase was purified to apparent homogeneity from cultured pheochromocytoma PC12 cells. The purified tumor enzyme was entirely dependent on tetrahydrobiopterin (BH4) for the hydroxylation of tyrosine to 3,4-dihydroxyphenylalanine and hydrogen peroxide could not substitute for the natural cofactor. Indeed, in the presence of BH4, increasing concentrations of hydrogen peroxide completely inhibited enzyme activity. The PC12 hydroxylase exhibited typical kinetics of tyrosine hydroxylation exhibited typical kinetics of tyrosine hydroxylation, both as a function of tyrosine (S0.5 Tyr = 15 microM) and BH4 (apparent Km BH4 = 210 microM). In addition, the enzyme catalyzed the hydroxylation of substantial amounts of phenylalanine to tyrosine and 3,4-dihydroxyphenylalanine (apparent Km Phe = 100 microM). Phenylalanine did not inhibit the enzyme in the concentrations tested, whereas tyrosine showed typical substrate inhibition at concentrations greater than or equal to 50 microM. At higher substrate concentrations, the rate of phenylalanine hydroxylation was equal to or exceeded that of tyrosine. Essentially identical results were obtained with purified tyrosine hydroxylase from pheochromocytoma PC18 cells. The data suggest that the tumor enzyme has the same substrate specificity and sensitivity to hydrogen peroxide as tyrosine hydroxylase from other tissues.     

Inhibition of Tyrosine Hydroxylase by R and S Enantiomers of Salsolinol, 1-Methyl-6,7-Dihydroxy-1,2,3,4- Tetrahydroisoquinoline

Salsolinol is one of the dopamine-derived tetrahydroisoquinolines and is synthesized from pyruvate or acetaldehyde and dopamine. As it cannot cross the blood-brain barrier, salsolinol as the R enantiomer in the brain is considered to be synthesized in situ in dopaminergic neurons. Effects of R and S enantiomers of salsolinol on kinetic properties of tyrosine hydroxylase [tyrosine, tetrahydrobiopterin:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2], the rate-limiting enzyme of catecholamine biosynthesis, were examined. The naturally occurring cofactor of tyrosine hydroxylase, L-erythro-5,6,7,8-tetrahydrobiopterin, was found to induce allostery to the enzyme polymers and to change the affinity to the biopterin itself. Using L-erythro-5,6,7,8-tetrahydrobiopterin, tyrosine hydroxylase recognized the stereochemical structures of the salsolinols differently. The asymmetric center of salsolinol at C-1 played an important role in changing the affinity to L-tyrosine. The allostery of tyrosine hydroxylase toward biopterin cofactors disappeared, and at low concentrations of biopterin such as in brain tissue, the affinity to the cofactor changed markedly. A new type of inhibition of tyrosine hydroxylase, by depleting the allosteric effect of the endogenous biopterin, was found. It is suggested that under physiological conditions, such a conformational change may alter the regulation of DOPA biosynthesis in the brain.     

Effects of melanin on tyrosine hydroxylase and phenylalanine hydroxylase.

Melanin inhibited rat liver phenylalanine hydroxylase, but activated tyrosine hydroxylase from rat brain (caudate nucleus), rat adrenal glands, and bovine adrenal medulla. Activation of tyrosine hydroxylase by melanin was demonstrated with the extensively dialyzed enzyme and in suboptimal concentrations of the substrate (tyrosine) and the cofactor (6-methyltetrahydropterin). Tyrosine hydroxylase from rat brain was activated by melanin more markedly than that from rat adrenal glands. Purified and extensively dialyzed bovine adrenal tyrosine hydroxylase had two Km values with 6-methyltetrahydropterin, depending upon its concentrations, but the melanin-activated tyrosine hydroxylase had a single Km value and showed the classical Michaelis-Menten kinetics.     

THE INHIBITION OF PHENYLALANINE AND TYROSINE HYDROXYLASES BY HIGH OXYGEN LEVELS

Abstract— The K _m for oxygen for rat liver phenylalanine hydroxylase depended on the structure of the reduced pterin cofactor. When the synthetic cofactor, 6,7-dimethyltetrahydropterin, was employed, the apparent K _m for oxygen was 20%. When the natural cofactor, tetrahydrobiopterin, was used, the apparent K _m for oxygen was 0.35 %. Substrate inhibition (40 per cent inhibition at 43% oxygen) was observed with the natural cofactor but not with the synthetic cofactor. Oxygen also caused substrate inhibition with bovine adrenal medulla and brain tyrosine hydroxylases. The inhibition was more dramatic in the presence of the natural cofactor than with the synthetic cofactor. Substrate inhibition by oxygen of brain tyrosine hydroxylase may explain the lowered brain levels of norepinephrine and dopamine observed after treatment of animals with hyperbaric oxygen.     

Immobilized phenylalanine hydroxylase through the SH groups

Purified rat liver phenylalanine hydroxylase [L-phenylalanine:tetrahydropteridine:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1] was immobilized with activated thiol-Sepharose 4B via disulfide bond formation, which is expected to immobilize the enzyme in its activated form through the SH modification. This immobilized enzyme was more stable against thermal denaturation than the free enzyme. When tetrahydrobiopterin was used as the natural cofactor, the K(m) value for phenylalanine was decreased and that for the cofactor was increased. Constant conversion from phenylalanine to tyrosine was demonstrated continuously for over 8 h at 25 degrees C.     

Adrenal tyrosine hydroxylase activation in the developing rat: influence of the thyroid status

Adrenal tyrosine hydroxylase activation was elicited in developing control, hypo- and hyperthyroid rats by insulin-hypoglycaemia. Rats were deeply anaesthetized with chloroform at a low concentration, since intrinsic tyrosine hydroxylase activation was very low with this technique, as compared to Ketamine injection or chloroform at a high concentration. The study of time-course of tyrosine hydroxylase activation showed that the maximum value was observed 2 h after insulin administration. In control animals, tyrosine hydroxylase activation increased between 4 and 20 days, and then decreased. Hypothyroidism is associated with a decreased tyrosine hydroxylase activation between 4 and 50 days, as compared to controls and hyperthyroidism with an increased activation between 6 and 30 days. While tyrosine hydroxylase from saline-treated rats exhibits two different forms (with two apparent Km values for the cofactor), enzyme from insulin-treated animals was present in a single form with a Km corresponding to the low Km value of the saline-injected rats. At 6 and 14 days, hypothyroidism increases tyrosine hydroxylase Km values as compared to euthyroid animals.     

Biopterin cofactor and monoamine-synthesizing monooxygenases

The activities of three pterin-requiring monooxygenases, phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase, are regulated by the level of the pterin cofactor, $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$, which is synthesized from guanosine triphosphate (GTP). Since tyrosine hydroxylase or tryptophan hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines (dopamine, norepinephrine and epinephrine) or serotonin in monoaminergic neurons, biosynthesis of tetrahydrobiopterin from GTP may also regulate the tissue level of monoamine transmitters. Recent evidences indicate that biosynthesis of tetrahydrobiopterin and that of biogenic monoamines may be regulated each other.     

Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases.

The chiral specificities of bovine striatal tyrosine hydroxylase (TH) (unphosphorylated and phosphorylated by cAMP-dependent protein kinase) and rat liver phenylalanine hydroxylase (PH) were examined at physiological pH using the pure C6 stereoisomers of 6-methyl- and 6-propyl-5,6,7,8-tetrahydropterin (6-methyl-PH4 and 6-propyl-PH4) and (6R)- and (6S)-tetrahydrobiopterin (BH4). Both PH and phosphorylated TH have substantially higher Vmax values with the unnatural (6R)-propyl-PH4 than the natural (6S)-propyl-PH4 (approximately 6- and 11-fold, respectively). However, the Km's are also higher such that Vmax/Km is almost unaffected by C6 chirality. Unphosphorylated TH has equal Km values for both isomers of 6-propyl-PH4, but has about a 6 times greater Vmax with the unnatural isomer, making it the fastest cofactor yet for this form of the enzyme. With the shorter 6-methyl group, chiral differences are still recognized by phosphorylated TH but hardly at all by PH. Inhibition of both PH and TH by amino acid substrate which occurs with (6R)-BH4 as cofactor is also observed with (6S)-propyl-PH4 but not with (6S)-BH4, (6R)-propyl-PH4, or (6R)- or (6R,S)-methyl-PH4. The Km for (6S)-BH4 with phosphorylated TH is nearly 3 times higher than with (6R)-BH4, but Vmax is unchanged. With unphosphorylated TH, (6S)-BH4 produces very low decelerating rates, which was shown not to be due to irreversible inactivation of the enzyme. The Km for (6R)-BH4 with either hydroxylase is 10 times higher than for the equivalently configured (6S)-propyl-PH4. Comparison of these two cofactors reveals that the 1' and 2' side-chain hydroxyl groups of the natural cofactor promote different regulatory functions in PH than in TH.     

Neurochemical Effects Following Peripheral Administration of Tetrahydropterin Derivatives to the hph-1 Mouse

Abstract: The hph-1 mouse, which displays tetrahydrobiopterin deficiency and impaired dopamine and serotonin turnover, has been used to study cofactor replacement therapy for disorders causing brain tetrahydrobiopterin deficiency. Subcutaneous administration of 100 µmol/kg (30 mg/kg) of tetrahydrobiopterin resulted in a twofold increase in brain cofactor concentration 1 h after administration. Concentrations remained above the endogenous level for at least 4 h but returned to normal by 24 h. The lipophilic tetrahydrobiopterin analogue 6-methyltetrahydropterin entered the brain five times more efficiently than tetrahydrobiopterin but was cleared at a faster rate. Tetrahydropterins linked to the lipoidal carrier N -benzyl-1,4-dihydronicotinoyl did not result in a detectable increase in levels of brain pterins over the period of the study (1–4 h). Stimulation of monoamine turnover was not observed at any time point with either natural cofactor or the methyl analogue. Increasing the amount of tetrahydrobiopterin to 1,000 µmol/kg resulted in elevation of cofactor concentrations, a brief increase in the activity of tyrosine and tryptophan hydroxylase 1 h postadministration, and increased turnover of dopamine and serotonin metabolites lasting 24 h. However, 2 of 12 (17%) mice died following administration of this dose of cofactor. Our findings suggest that acute peripheral tetrahydrobiopterin administration is unlikely to stimulate brain monoamine turnover directly unless very large and potentially toxic doses of cofactor are used.     

Direct binding of GTP cyclohydrolase and tyrosine hydroxylase: regulatory interactions between key enzymes in dopamine biosynthesis

The signaling functions of dopamine require a finely tuned regulatory network for rapid induction and suppression of output. A key target of regulation is the enzyme tyrosine hydroxylase, the rate-limiting enzyme in dopamine synthesis, which is activated by phosphorylation and modulated by the availability of its cofactor, tetrahydrobiopterin. The first enzyme in the cofactor synthesis pathway, GTP cyclohydrolase I, is activated by phosphorylation and inhibited by tetrahydrobiopterin. We previously reported that deficits in GTP cyclohydrolase activity in Drosophila heterozygous for mutant alleles of the gene encoding this enzyme led to tightly corresponding diminution of in vivo tyrosine hydroxylase activity that could not be rescued by exogenous cofactor. We also found that the two enzymes could be coimmunoprecipitated from tissue extracts and proposed functional interactions between the enzymes that extended beyond provision of cofactor by one pathway for another. Here, we confirm the physical association of these enzymes, identifying interacting regions in both, and we demonstrate that their association can be regulated by phosphorylation. The functional consequences of the interaction include an increase in GTP cyclohydrolase activity, with concomitant protection from end-product feedback inhibition. In vivo, this effect would in turn provide sufficient cofactor when demand for catecholamine synthesis is greatest. The activity of tyrosine hydroxylase is also increased by this interaction, in excess of the stimulation resulting from phosphorylation alone. Vmax is elevated, with no change in Km. These results demonstrate that these enzymes engage in mutual positive regulation.     

Nanosecond pulse fluorometry of conformational change in phenylalanine hydroxylase associated with activation

Conformational change in rat liver phenylalanine hydroxylase associated with activation by phenylalanine or N-(1-anilinonaphth-4-yl)maleimide was investigated by measuring fluorescence spectra and fluorescence lifetimes of tryptophanyl residues as well as the probe fluorophore conjugated with SH groups of the hydroxylase. The fluorescence spectrum of tryptophan exhibited its maximum at 342 nm. It shifted by 8 nm toward longer wavelength accompanied by an increase in its intensity, by preincubation with 1 mM phenylalanine. The fluorescence intensity of tryptophan increased by 36% upon the activation. On the other hand, the binding of (6R)-L-erythro-tetrahydrobiopterin, a natural cofactor of the enzyme, induced a decrease in the fluorescence intensity by 79% without a shift of the maximum wavelength. The fluorescence lifetime of tryptophan of phenylalanine hydroxylase exhibited two components with lifetimes of 1.7 and 4.1 ns. The values of the lifetimes changed to 1.4 and 5.6 ns, respectively, upon the activation. It is considered that the change in the longer lifetime is correlated with the shift of the emission peak upon the activation. The values of both the lifetimes decreased to 0.64 and 3.6 ns upon the binding of (6R)-L-erythro-tetrahydrobiopterin, which is coincident with the decrease in the fluorescence intensity. Conjugation of N-(1-anilinonaphth-4-yl)maleimide with SH of phenylalanine hydroxylase brought about a decrease in both the fluorescence intensity and the value of the shorter lifetime of the tryptophanyl residues, while the longer lifetime remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and optimization of tyrosine hydroxylase activity in Mucuna pruriens DC. var. utilis

Tyrosine hydroxylase, an iron containing tetrahydrobiopterin dependent monooxygenase (tyrosine 3-monooxygenase; EC 1.14.16.2), catalyzes the rate-limiting step in which l-dopa is formed from the substrate l-tyrosine. l-Dopa concentration and activity of l-tyrosine hydroxylase enzyme were measured in roots, stem, leaves, pods, and immature seeds of Mucuna pruriens. Immature seeds contained maximum l-dopa content and mature leaves possessed maximum catalytic activity of tyrosine hydroxylase. Tyrosine hydroxylase from leaf homogenate was characterized as a 55 kDa protein by SDS-PAGE and Western-blot analysis with monoclonal mouse IgG2a tyrosine hydroxylase antibody. The conditions for maximum tyrosine hydroxylase activity from the leaf extract were optimized with respect to temperature, pH, cofactor 6-MPH₄, and divalent metal ions. The tyrosine hydroxylase from leaf extract possessed a K _m value of 808.63 μM for l-tyrosine at 37°C and pH 6.0. The activity of the enzyme was slightly inhibited at 2,000 μM l-tyrosine. Higher concentrations of the cofactor 6-MPH₄, however, completely inhibited the synthesis of l-dopa. Tyrosine hydroxylase converted specific monophenols such as l-tyrosine (808.63 μM) and tyramine (K _m 1.1 mM) to diphenols l-dopa and dopamine, respectively. Fe(II) activated the enzyme while higher concentration of other divalent metals reduced its activity. For the first time, tyrosine hydroxylase from M. pruriens is being reported in this study.     

Changes in the cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues

The structure of the cofactor binding domain of tyrosine hydroxylase (TH) was examined at physiological pH by determining kinetic parameters of (R)-tetrahydrobiopterin [(R)-BH4] and a series of tetrahydropterin (PH4) derivatives (6-R1-6-R2-PH4: R1 = H and R2 = methyl, hydroxymethyl, ethyl, methoxymethyl, phenyl, and cyclohexyl; R1 = methyl and R2 = methyl, ethyl, propyl, phenyl, and benzyl). A minimally purified TH preparation that was not specifically phosphorylated (designated as "unphosphorylated") was compared with enzyme phosphorylated with cAMP-dependent protein kinase. The Km for tyrosine with most tetrahydropterin analogues ranged between 20 and 60 microM with little decrease upon phosphorylation. Two exceptions were an unusually low Km of 7 microM with 6-ethyl-PH4 and a high Km of 120 microM with 6-phenyl-6-methyl-PH4, both with phosphorylated TH. Tyrosine substrate inhibition was elicited only with (R)-BH4 and 6-hydroxymethyl-PH4. With unphosphorylated TH (with the exception of 6-benzyl-6-methyl-PH4, Km = 4 mM) an inverse correlation between cofactor Km and side-chain hydrophobicity was observed ranging from a high with (R)-BH4 (5 mM) to a low with 6-cyclohexyl-PH4 (0.3 mM). An 8-fold span of Vmax was seen overall. Phosphorylation caused a 0.6-4-fold increase in Vmax and a 35-2000-fold decrease in Km for cofactor, ranging from a high of 60 microM with 6-methyl-PH4 to a low of 0.6 microM with 6-cyclohexyl-PH4. A correlation of the size of the hydrocarbon component of the side chain with affinity is strongly evident with phosphorylated TH, but in contrast to unphosphorylated enzyme, the hydroxyl groups in hydroxymethyl-PH4 (20 microM) and (R)-BH4 (3 microM) decrease Km in comparison to that of 6-methyl-PH4. Although 6,6-disubstituted analogues were found with affinities near that of (R)-BH4 (e.g., 6-propyl-6-methyl-PH4, 4 microM), they were frequently more loosely associated with phosphorylated TH than their monosubstituted counterparts (6-phenyl-PH4, 0.8 microM; cf. 6-phenyl-6-methyl-PH4, 8 microM). A model of the cofactor side-chain binding domain is proposed in which a limited region of nonpolar protein residue(s) capable of van der Waals contact with the hydrocarbon backbone of the (R)-BH4 dihydroxypropyl group is opposite to a recognition site for hydroxyl(s). Although interaction with either the hydrophilic or hydrophobic regions of unphosphorylated tyrosine hydroxylase is possible, phosphorylation by cAMP-dependent protein kinase appears to optimize the simultaneous operation of both forces.     

Modulation by pterins of the phosphorylation and phenylalanine activation of phenylalanine 4-mono-oxygenase.

The interaction between phenylalanine 4-mono-oxygenase and analogues of the natural cofactor (6R)-tetrahydrobiopterin [(6R)-BH4] was studied. The rate of cyclic AMP-dependent phosphorylation of phenylalanine 4-mono-oxygenase was inhibited only by those pterins [(6R)-BH4, (6S)-BH4 and 7,8-dihydrobiopterin (BH2)] that were able to decrease the potency and efficiency of phenylalanine as an allosteric activator of the hydroxylase. Since BH2 lacks cofactor activity, this was not required to modulate either the phosphorylation or the phenylalanine-activation of the hydroxylase. Half-maximal inhibition of the phosphorylation was observed at 1.9 microM-(6R)-BH4, 9 microM-(6S)-BH4 and 17 microM-BH2. Competition experiments indicated that all three pterins acted through binding to the cofactor site of the hydroxylase. Since the phosphorylation site and the cofactor binding site are known to reside, respectively, in the N- and C-terminal domains of the hydroxylase, the pterins were able to induce an interdomain conformational change. BH2, whose dihydroxypropyl group is not subject to epimerization, and (6S)-BH4 both inhibited the phosphorylation less efficiently than did the (6R)-epimer of BH4. Pterins with different spatial arrangements of the dihydroxypropyl side chain thus appeared to elicit different conformations of the phosphorylation site. The hydroxylase reaction showed a higher apparent Km for (6S)-BH4 than for (6R)-BH4 both when the native and the phenylalanine-activated enzyme were tested. For the activated enzyme Vmax was 40% lower with the (6S)-epimer than the (6R)-epimer, also when the more rapid enzyme inactivation occurring with the former cofactor was taken into account.