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.     

Activation of neuronal nitric-oxide synthase by the 5-methyl analog of tetrahydrobiopterin. Functional evidence against reductive oxygen activation by the pterin cofactor.

Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 microM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mM of the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.     

Inactivation of Brain Tryptophan Hydroxylase by Nitric Oxide

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by nitric oxide (NO) and by the NO generators sodium nitroprusside, diethylamine/NO, S -nitroso- N -acetylpenicillamine, and S -nitrosocysteine. The inactivation occurs in an oxygen-free environment and is enhanced by dithiothreitol and ascorbic acid. Protection against the effect of NO on tryptophan hydroxylase is afforded by oxyhemoglobin, reduced glutathione, and exogenous Fe(II). Catalase partially protects the enzyme from NO-induced inactivation, whereas both superoxide dismutase and uric acid are without effect. These findings indicate that tryptophan hydroxylase is a target for NO and suggest that critical iron-sulfur groups in this enzyme serve as the substrate for NO-induced nitrosylation of the protein, resulting in enzyme inactivation.     

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.     

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.     

Kinetic properties of tyrosine hydroxylase with natural tetrahydrobiopterin as cofactor

A reproducible purification procedure of native tyrosine hydroxylase (L-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the soluble fraction of the bovine adrenal medulla has been established. This procedure accomplished a 90-fold purification with a recovery of 30% of the activity. This purified enzyme served for studying the kinetic properties of tyrosine hydroxylase using (6R)-L-erythro-1',2'-dihydroxypropyltetrahydropterin [(6R)-L-erythro-tetrahydrobiopterin] as cofactor, which is supposed to be a natural cofactor. Two different Km values for tyrosine, oxygen and natural (6R)-L-erythro-tetrahydrobiopterin itself were obtained depending on the concentration of the tetrahydrobiopterin cofactor. In contrast, when unnatural (6S)-L-erythro-tetrahydrobiopterin was used as cofactor, a single Km value for each tyrosine, oxygen and the cofactor was obtained independent of the cofactor concentration. The lower Km value for (6R)-L-erythro-tetrahydrobiopterin was close to the tetrahydrobiopterin concentration in tissue, indicating a high affinity of the enzyme to the natural cofactor under the in vivo conditions. Tyrosine was inhibitory at 100 microM with (6R)-L-erythro-tetrahydrobiopterin as cofactor, and the inhibition by tyrosine was dependent on the concentrations of both pterin cofactor and oxygen. Oxygen at concentrations higher than 4.8% was also inhibitory with (6R)-L-erythro-tetrahydrobiopterin as cofactor.     

Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase.

Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin.     

L-ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin

Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of tetrahydrobiopterin. This was evident as an increase in the half-life of tetrahydrobiopterin in aqueous solution. Furthermore, the increase of tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.     

7-substituted pterins in humans with suspected pterin-4a-carbinolamine dehydratase deficiency. Mechanism of formation via non-enzymatic transformation from 6-substituted pterins.

A recently described new form of hyperphenylalaninemia is characterized by the excretion of 7-substituted isomers of biopterin and neopterin and 7-oxo-biopterin in the urine of patients. It has been shown that the 7-substituted isomers of biopterin and neopterin derive from L-tetrahydrobiopterin and D-tetrahydroneopterin and are formed during hydroxylation of phenylalanine to tyrosine with rat liver dehydratase-free phenylalanine hydroxylase. We have now obtained identical results using human phenylalanine hydroxylase. The identity of the pterin formed in vitro and derived from L-tetrahydrobiopterin as 7-(1',2'-dihydroxypropyl)pterin was proven by gas-chromatography mass spectrometry. Tetrahydroneopterin and 6-hydroxymethyltetrahydropterin also are converted to their corresponding 7-substituted isomers and serve as cofactors in the phenylalanine hydroxylase reaction. Dihydroneopterin is converted by dihydrofolate reductase to the tetrahydro form which is biologically active as a cofactor for the aromatic amino acid monooxygenases. The 6-substituted pterin to 7-substituted pterin conversion occurs in the absence of pterin-4a-carbinolamine dehydratase and is shown to be a nonenzymatic process. 7-Tetrahydrobiopterin is both a substrate (cofactor) and a competitive inhibitor with 6-tetrahydrobiopterin (Ki approximately 8 microM) in the phenylalanine hydroxylase reaction. For the first time, the formation of 7-substituted pterins from their 6-substituted isomers has been demonstrated with tyrosine hydroxylase, another important mammalian enzyme which functions in the hydroxylation of phenylalanine and tyrosine.     

Dopamine-Releasing Action of 6R-l-erythro-Tetrahydrobiopterin: Analysis of Its Action Site Using Sepiapterin

Abstract: Recently, we reported that 6 R - l - erythro -tetrahydrobiopterin (6 R -BH₄), a natural cofactor for hydroxylases of tyrosine and tryptophan, has a monoamine-releasing action independent of its cofactor activity. Here we attempted to determine whether 6 R -BH₄ acts inside the cell or from the outside of the cell by using brain microdialysis in the rat striatum. For this purpose, sepiapterin, an immediate precursor of 6 R -BH₄ in the salvage pathway, was used to selectively increase the intracellular 6 R -BH₄ levels. Dialytic perfusion of sepiapterin increased tissue levels of reduced biopterin (mainly 6 R -BH₄) but not the extracellular levels. Administration of sepiapterin increased the extracellular levels of 3,4-dihydroxyphenylalanine (DOPA) (an index of in vivo tyrosine hydroxylase activity) and of dopamine (DA) (an index of in vivo DA release). Either of the increases was eliminated after pretreatment with a tyrosine hydroxylase inhibitor α-methyl- p -tyrosine. Administration of 6 R -BH₄ increased extracellular levels of reduced biopterin, DOPA, and DA. After pretreatment with α-methyl- p -tyrosine, the increase in DOPA levels was abolished, but most of the increase in DA levels persisted. The increase in DA levels also persisted after pretreatment with nitric oxide synthase inhibitors. These data demonstrate that 6 R -BH₄ stimulates DA release directly, independent of its cofactor action for tyrosine hydroxylase and nitric oxide synthase, by acting from the outside of neurons.     

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.     

Stability Properties of Activated Tryptophan Hydroxylase from Rat Midbrain

Abstract: Time courses of the activation-inactivation sequence in rat midbrain tryptophan hydroxylase after preincubation with calcium, ATP + MgCl₂, or sulfhydryl reagents and after freezing and thawing suggest that the activated enzyme is more vulnerable to loss of activity. The sequence induced by calcium was prevented by the protease inhibitor leupeptin, and an accelerated decline in activity after activation by ATP + MgCl₂ was reduced greatly by increasing levels of tetrahydrobiopterin (BH₄) cofactor. The effects of calcium and ATP + MgCl₂ were additive, which suggests independent mechanisms. The findings suggest that time courses of enzyme activation and inactivation processes may offer a useful way to study the influence of a range of effectors on tryptophan hydroxylase function.     

Feedback Inhibition of Tyrosine Hydroxylase from Five Regions of Rat Brain by 2-Hydroxyestradiol and Dihy droxy pheny lalanine

Abstract: Inhibition of tyrosine hydroxylase from five regions of rat brain by a model catecholamine, DOPA and a model catecholestrogen, 2-hydroxyestradiol, was examined. Tyrosine hydroxylase preparations from amygdala, preoptic, hypothalamic, striatal and hippocampal regions were freed of small molecules by gel filtration before use. The feedback inhibition of tyrosine hydroxylase by the two model catechols was studied using a spectrum of reduced pterin cofactors, including tetrahydrobiopterin, 6-methyl-tetrahydropterin and 6, 7-dimethyltetrahydropterin. Micromolar levels of either inhibitor produced marked inhibition of tyrosine hydroxylase from all regions when subsaturating levels of the endogenous cofactor, tetrahydrobiopterin, were used.     

Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein

GTP cyclohydrolase I (GTPCHI) is the rate-limiting enzyme involved in the biosynthesis of tetrahydrobiopterin, a key cofactor necessary for nitric oxide synthase and for the hydroxylases that are involved in the production of catecholamines and serotonin. In animals, the GTPCHI feedback regulatory protein (GFRP) binds GTPCHI to mediate feed-forward activation of GTPCHI activity in the presence of phenylalanine, whereas it induces feedback inhibition of enzyme activity in the presence of biopterin. Here, we have reported the crystal structure of the biopterin-induced inhibitory complex of GTPCHI and GFRP and compared it with the previously reported phenylalanine-induced stimulatory complex. The structure reveals five biopterin molecules located at each interface between GTPCHI and GFRP. Induced fitting structural changes by the biopterin binding expand large conformational changes in GTPCHI peptide segments forming the active site, resulting in inhibition of the activity. By locating 3,4-dihydroxy-phenylalanine-responsive dystonia mutations in the complex structure, we found mutations that may possibly disturb the GFRP-mediated regulation of GTPCHI.     

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.     

Studies on the phenylalanine hydroxylase system in liver slices.

A method was developed to study the unsupplemented phenylalanine hydroxylase system in rat liver slices. All of the components of the system--tetrahydrobiopterin, dihydropteridine reductase, and the hydroxylase itself--are present under conditions which should be representative of the actual physiological state of the animal. The properties of the system in liver slices have been compared to those of the purified enzyme in vitro. The three pterins, tetrahydrobiopterin, 6,7-dimethyltetrahydropterin, and 6-methyltetrahydropterin, all stimulate the hydroxylation of phenylalanine when added to the liver slice medium in the presence of a chemical reducing agent. The relative velocities found at 1 mM phenylalanine and saturating pterin concentrations are: tetrahydrobiopterin, 1; 6,7-dimethyltetrahydropterin, 2.5; 6-methyltetrahydropterin, 13. This ratio of activities is similar to that found for the purified, native phenylalanine hydroxylase and indicates that the enzyme in vivo is predominantly in the native form. Rats pretreated with 6-methyltetrahydropterin showed enhanced phenylalanine hydroxylase activity in liver slices demonstrating for the first time that an exogenous tetrahydropterin can interact with the phenylalanine hydroxylase system in vivo. This finding opens up the possibility of treating phenylketonurics who still possess some residual phenylalanine hydroxylase activity with a tetrahydropterin like 6-methyltetrahydropterin which can give a large increase in rate over that seen with the natural cofactor, tetrahydrobiopterin.     

Evidence for the formation of the 4a-carbinolamine during the tyrosine-dependent oxidation of tetrahydrobiopterin by rat liver phenylalanine hydroxylase

In the presence of phenylalanine and molecular oxygen, activated phenylalanine hydroxylase catalyzes the oxidation of tetrahydrobiopterin. The oxidation of this tetrahydropterin cofactor also proceeds if the substrate, phenylalanine, is replaced by its product, tyrosine, in the initial reaction mixture. These two reactions have been defined as coupled and uncoupled, respectively, because in the former reaction 1 mol of phenylalanine is hydroxylated for every mole of tetrahydrobiopterin oxidized, whereas in the latter reaction there is no net hydroxylation of tyrosine during the oxidation of the tetrahydropterin. During the course of the coupled oxidation of tetrahydrobiopterin, a pterin 4a-carbinolamine intermediate can be detected by ultraviolet spectroscopy (Kaufman, S. (1976) in Iron and Copper Proteins (Yasunobu, K. T., Mower, H. F., and Hayaishi, O., eds) pp. 91-102, Plenum Publishing Corp., New York). Dix and Benkovic (Dix, T. A., and Benkovic, S. J. (1985) Biochemistry 24, 5839-5846) have postulated that the formation of this intermediate only occurs when the oxidation of the tetrahydropteridine is tightly coupled to the concomitant hydroxylation of the aromatic amino acid. However, during the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase, we have detected the formation of a spectral intermediate with ultraviolet absorbance that is essentially identical to that of the carbinolamine. Furthermore, this absorbance can be eliminated by the addition of 4a-carbinolamine dehydratase, an enzyme which catalyzes the dehydration of the 4a-carbinolamine. Quantitation of this intermediate suggests that there are two pathways for the tyrosine-dependent uncoupled oxidation of tetrahydrobiopterin by phenylalanine hydroxylase because only about 0.3 mol of the intermediate is formed per mol of the cofactor oxidized.     

Effects of thyrotropin releasing hormone and its analogue, DN 1417, on biopterin concentration and tryptophan hydroxylation in rat raphe slices

The effects of subchronic administration of thyrotropin releasing hormone (TRH) and its analogue, gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN 1417), on serotonin biosynthesis in situ were investigated in tissue slices of the midbrain raphe of rats. TRH or DN 1417 (10 mg/kg per day intraperitoneally) were administered to male Wistar rats for ten days. At twenty four hr after the last injection, tissue slices of the midbrain raphe were prepared and the rate of serotonin biosynthesis was estimated by measuring formation of 5-hydroxytryptophan (5-HTP) from tryptophan during inhibition of aromatic L-amino acid decarboxylase using high-performance liquid chromatography with fluorescence detection. Total biopterin content was determined by a specific radioimmunoassay. 5-HTP formation was decreased 22% and 29%, and total biopterin content 69% and 72%, in TRH- and DN 1417-treated rats, respectively. However, tryptophan concentration in raphe slices did not change. In contrast, the Vmax of tryptophan hydroxylase in the homogenate of the raphe nucleus in the presence of a saturating concentration of (6R)-L-erythro-tetrahydrobiopterin, the naturally occurring pterin cofactor, was significantly increased after repeated administration of TRH or DN 1417. These results indicate that reduction of in situ serotonin biosynthesis in tissue slices from the rats treated with TRH or DN 1417 subchronically contray to the increase in in vitro tryptophan hydroxylase may result from the decrease of the biopterin cofactor, and that changes in concentrations of the biopterin cofactor may play a regulatory role in serotonin biosynthesis in vivo under certain conditions.     

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.     

Intracerebrally administered (6r)-l-erythro-tetrahydrobiopterin does not affect extracellular levels of dopamine and serotonin metabolites in rat striatum in vivo during measurement by brain micro-dialysis system

By the use of the brain micro-dialysis technique combined with HPLC, the changes in the extracellular levels of dopamine (DA) and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), and a serotonin(5-HT) metabolite, 5-hydroxyindoleacetic acid (5-HIAA) were examined in the rat striatum before and after intracerebral injection of a vehicle or (6R)-l-erythro-tetrahydrobiopterin (6R-BH₄), the natural form of the cofactor for the tryrosine hydroxylase and tryptophan hydroxylase. No apparent change after the 6R-BH, treatment was found in the levels of DA, DOPAC, HVA and 5-HIAA in the striatal dialysate. In contrast, the levels of total biopterin in both the operated (dialysis probe-implanted) and unoperated striatum of 6R-BH₄-treated rats increased by 23- and 93-fold, respectively, when compared with those of the control, vehicle-treated rats. The results indicate that increased levels of the tetrahydrobiopterin cofactor may not affect the release of DA and the extracellular level of DA and 5-HT metabolites in the physiologically normal brain.