Formation of Catechol Compounds from Phenylalanine and Tyrosine with Isolated Nerve Endings

THERE is much evidence that catecholamines may act as synaptic transmitters in the mammalian brain¹. Enzymatic activities necessary for the synthesis of catecholamines have been located in central neurones¹ and it is generally believed that tyrosine hydroxylase² is the rate limiting enzyme in brain as well as peripheral tissues containing catecholamines³. While it is clear that tyrosine can serve as a precursor of catecholamine synthesis in the brain^{1, 3, 4}, the significance of phenylalanine is problematic. It was believed that the mammalian brain is devoid of enzymatic activity necessary to convert phenylalanine to tyrosine^{6, 7}, while liver is known to be rich in the enzyme phenylalanine hydroxylase⁸. The earlier attempts to demonstrate hydroxylation of phenylalanine in brain tissue may have been unsuccessful due to methodological problems⁹. Recent evidence suggests that tyrosine hydroxylase prepared from peripheral sympathetically innervated tissues or from brain can hydroxylate either phenylalanine or tyrosine⁹. Initially, the rate of hydroxylation of phenylalanine by tyrosine hydroxylase was thought to be as little as 5% that of tyrosine⁹. It has been found recently, however, that structural variations in the pteridine cofactor present in the incubation mixture lead to striking changes in the ability of partially purified tyrosine hydroxylase from bovine adrenal medulla to hydroxylate phenylalanine¹⁰. Thus, tetrahydrobiopterin allowed the hydroxylation of phenylalanine to proceed at least as rapidly as that of tyrosine or faster¹⁰. As the structure of the endogenous pteridine cofactor of tyrosine hydroxylase is not known, it is possible that synthesis of catecholamines from phenylalanine as well as tyrosine could occur in intact neuronal tissues. Evidence has been presented that after the injection of large quantities of ¹⁴C-phenylalanine into the lateral ventricle of the rat brain, small amounts of labelled tyrosine and traces of newly synthesized catecholamines were detected in brain tissues, giving qualitative evidence that catecholamines may be synthesized in brain from phenylalanine in vivo¹¹.     

The role of reductants in the tyrosine hydroxylase reaction.

The role of several reducing systems in the tyrosine hydroxylase reaction has been studied. A significant dependence upon the reducing systems beyond that required to regenerate the oxidized cofactor has been observed. 2-Mercaptoethanol, NADPH, and ascorbate are each effective at reducing the cofactor, but their abilities to stimulate tyrosine hydroxylase vary over a threefold range. NADPH is a suitable reductant for the tyrosine hydroxylase reaction, even in the absence of pteridine reductase. A reducing system containing ascorbate, ferrous ion, and catalase gives unusually high enzyme activity and low blanks. This ascorbate system, in addition to being useful for in vitro enzyme assays, may serve as a model for the in vivo reaction. Ascorbate may play an important role in the hydroxylation of tyrosine in catecholaminergic tissues. This study demonstrates that an efficient reductant for the tyrosine hydroxylase reaction must, in addition to reducing the pterin cofactor, also interact effectively with the enzyme itself.     

Short-term effect of stress on tyrosine hydroxylase activity

The short-term influences of stress on the activities of tyrosine hydroxylase in vivo and in vitro were examined in mice. The in vivo tyrosine hydroxylase activity was estimated by the rate of dopa accumulation which was measured at 30 min after the injection of NSD-1015 (100 mg kg), an aromatic l-amino acid decarboxylase inhibitor, intraperitoneally and was compared with tyrosine hydroxylase activity measured in vitro. For the in vivo assay, both the accumulation of dopa (tyrosine hydroxylase activity) and that of 5-hydroxytryptophan (tryptophan hydroxylase activity) and the levels of monoamines and the metabolites (noradrenalin, adrenalin, dopamine, normetanephrine, 3-methoxytyramine and serotonin) and those of precursor amino acids, tyrosine and tryptophan, were investigated in ten different brain regions and in adrenals. The amount of dopa accumulation in the brain as a consequence of decarboxylase inhibition, in vivo tyrosine hydroxylase activity, was significantly increased by stress, in nerve terminals (striatum, limbic brain, hypothalamus, cerebral cortex and cerebellum) and also in adrenals. The effect of stress on tyrosine hydroxylase activity in vitro at a subsaturating concentration of 6-methyltetrahydropterin cofactor was also observed in nerve terminals (striatum, limbic brain, hypothalamus, and cerebral cortex). The amount of 5-hydroxytryptophan accumulation, the in vivo tryptophan hydroxylase activity, was also significantly increased in bulbus olfactorius, limbic brain, cerebral cortex, septum and lower brain stem. The influence of stress was also observed on the levels of precursor amino acids, tyrosine and tryptophan and monoamines in specific brain parts. These results suggest that the stress influences both catecholaminergic neurons and serotonergic neurons in nerve terminals in the brain. This effect was also observed on tyrosine hydroxylase activity in vitro in nerve terminals. However, in adrenals, the influence by stress was not observed on the in vitro activity, although dopa accumulation was increased.     

A comparative study of Drosophila phenylalanine hydroxylase with a natural and a synthetic tetrahydropterin as cofactor.

1. Phenylalanine hydroxylase activity has been analyzed in Drosophila melanogaster using as cofactors the natural tetrahydropteridine 5,6,7,8-tetrahydrobiopterin (H4Bip) and the synthetic one 5,6-dimethyl-5,6,7,8-tetrahydropterin (H4Dmp). 2. The apparent Vmax and KM for substrate and cofactor showed that the enzyme has two times more affinity for the substrate when H4Bip is the cofactor in the reaction. Similarly to what was found with purified rat liver phenylalanine hydroxylase, H4Bip was the most effective cofactor, leading to 4-5 times more activity than that obtained with H4Dmp. 3. With the natural cofactor H4Bip, no activation of the enzyme with Phe was necessary (in contrast to mammalian phenylalanine hydroxylase), and this tetrahydropteridine inhibits phenylalanine hydroxylase activity when the enzyme is exposed to it before phenylalanine addition. With the synthetic H4Dmp, both types of preincubations led to an increase of phenylalanine hydroxylase activity. 4. The enzyme is highly unstable compared to mammalian phenylalanine hydroxylase, even at -20 degrees C. 5. Thorax and abdomen extracts caused significant inhibition of phenylalanine hydroxylase activity from third instar larvae or newborn adult head extracts, when assayed with the synthetic cofactor H4Dmp. This inhibition did not happen with H4Bip. The presence of the pteridine 7-xanthopterin in adult bodies was not the cause of this inhibition.     

In vitro effects of pH and phosphorylation on neostriatal tyrosine hydroxylase from control and haloperidol-treated rats.

Acute administration of haloperidol to rats causes a marked decrease in the Km of neostriatal tyrosine hydroxylase for the pteridine cofactor, 6MPH₄, with no change in Vmax. We report that this effect is dependent on the pH of the assay mixture. It occurs at pH 6.5 but not at pH 6.0, the pH optimum for TH. With phosphorylating conditions at pH 6.5, the haloperidol-induced activation is no longer observed and the kinetics of TH are the same as those from control rats. At pH values of 6.0, 6.3 and 6.5, a significant decrease in Vmax occurs, with increasing pH, while no significant change in Km for the cofactor is observed for TH from control rats. However, when phosphorylating conditions are employed a marked increase in Km for the cofactor is observed while only a slight decrease in Vmax is seen, with increasing pH, for the control enzyme at the three pH values tested.     

Pyrimidines as cofactors for phenylalanine hydroxylase

It has been generally assumed that a tetrahydropterin (2-amino-5,6,7,8-tetrahydro-4-pteridinone) is essential for activity of the three aromatic amino acid hydroxylases. In this report it is shown that appropriately substituted pyrimidines can assume the role of cofactor for phenylalanine hydroxylase. 2,5,6-Triamino-4-pyrimidinone(V) and 5-benzylamino-2,6-diamino-4-pyrimidinone(VI) possess the same K_m values (0.1 mM and 0.003 mM) and stoichiometry of tyrosine generated to cofactor consumed (0.4 and 1.0) as their corresponding pteridine analogs, tetrahydropterin(III) and 6-phenyltetrahydropterin(IV). However, the rates with pyrimidines are lower. The ratio of rates $\text{V}\text{III}\text{= 0.045}$ and $\text{VI}\text{IV}\text{= 0.015}$. These results indicate that pteridine carbons 6 and 7 are not fundamental to cofactor binding or function, though they markedly influence the maximum velocity of hydroxylation. Pyrimidine cofactors of phenylalanine hydroxylase are valuable probes for the elucidation of the binding forces, transition states, and mechanism of oxygen activation of these hydroxylases.     

Activation of snail (Helix pomatia) nervous tissue tyrosine monooxygenase by calcium in vitro.

Addition of calcium chloride to soluble preparations of tyrosine monooxygenase from snail brain appears to produce an activation of the enzyme when assayed with subsaturating concentrations of the pteridine cofactor 6 MPH4 (2-amino-4-hydroxy-6-methyltetrahydropteridine). While some increase in the activity occurs with calcium chloride at a concentration of 0.01 mM, activation is increased by about 100% at 1mM and reaches a maximum at 5mM (144%) where it remains more or less constant up to 10mM. Barium chloride also produces an activating effect although it is much less pronounced while magnesium chloride is without effect. EGTA has no direct effect on the enzyme but antagonises the activation produced by calcium chloride. The activation of tyrosine monooxygenase by calcium is reflected in changes in the kinetic properties of the enzyme, decreasing the Km from 43 muM to 19 muM for tyrosine and from 670muM to 230muM for the pteridine cofactor. No change was observed with V values for either tyrosine or pteridine cofactor. It is suggested that calcium, which enters the nerve terminal during nerve stimulation, regulates the transmitter dopamine by activating the rate-limiting enzyme tyrosine monooxygenase.     

Interaction of 5-methyltetrahydrofolate and tetrahydrobiopterin on endothelial function

The present study was designed to investigate the interaction between 5-methyltetrahydrofolate and tetrahydrobiopterin in modulating endothelial function. Tetrahydrobiopterin is a critical cofactor for nitric oxide synthase and maintains this enzyme as a nitric oxide- versus superoxide-producing enzyme. The structure of 5-methyltetrahydrofolate is similar to tetrahydrobiopterin and both agents have been shown to improve endothelium-dependent vasodilatation. We hypothesized that 5-methyltetrahydrofolate interacts with nitric oxide synthase in a fashion analogous, yet independent, of tetrahydrobiopterin to improve endothelial function. We demonstrate that 5-methyltetrahydrofolate binds the active site of nitric oxide synthase and mimics the orientation of tetrahydrobiopterin. Furthermore, 5-methyltetrahydrofolate attenuates superoxide production (induced by inhibition of tetrahydrobiopterin synthesis) and improves endothelial function in aortae isolated from tetrahydrobiopterin-deficient rats. We suggest that 5-methyltetrahydrofolate directly interacts with nitric oxide synthase to promote nitric oxide (vs. superoxide) production and improve endothelial function. 5-Methyltetrahydrofolate may represent an important strategy for intervention aimed at improving tetrahydrobiopterin bioavailability.     

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.     

Tyrosine hydroxylase: mechanisms of oxygen radical formation

Summary

A new mechanism of oxygen radical formation in dopaminergic neurons is proposed, based on the oxidative mechanism of tyrosine hydroxylase. The cofactor (6R,6S)-5,6,7,8-tetrahydrobiopterin can rearrange in solution which allows an autoxidation reaction producing O₂^.-, H₂O₂ and HO^.. The combination of tyrosine hydroxylase and the cofactor produces more oxygen radicals than does the autoxidation of the cofactor. This production of oxygen radicals could be damaging to dopaminergic neurons. In the presence of tyrosine, the enzyme produces less radicals than it does in the absence of tyrosine. Mechanisms are proposed for the generation of reactive oxygen species during the autoxidation of the cofactor and during enzymatic catalysis. The generation, by tyrosine hydroxylase, of very small amounts of oxygen radicals over the period of 65 years could contribute to the oxidative stress that causes Parkinson's disease.     

Endogenous tetrahydroisoquinolines associated with Parkinson's disease mimic the feedback inhibition of tyrosine hydroxylase by catecholamines

N-methyl-norsalsolinol and related tetrahydroisoquinolines accumulate in the nigrostriatal system of the human brain and are increased in the cerebrospinal fluid of patients with Parkinson's disease. We show here that 6,7-dihydroxylated tetrahydroisoquinolines such as N-methyl-norsalsolinol inhibit tyrosine hydroxylase, the key enzyme in dopamine synthesis, by imitating the mechanisms of catecholamine feedback regulation. Docked into a model of the enzyme's active site, 6,7-dihydroxylated tetrahydroisoquinolines were ligated directly to the iron in the catalytic center, occupying the same position as the catecholamine inhibitor dopamine. In this position, the ligands competed with the essential tetrahydropterin cofactor for access to the active site. Electron paramagnetic resonance spectroscopy revealed that, like dopamine, 6,7-dihydroxylated tetrahydroisoquinolines rapidly convert the catalytic iron to a ferric (inactive) state. Catecholamine binding increases the thermal stability of tyrosine hydroxylase and improves its resistance to proteolysis. We observed a similar effect after incubation with N-methyl-norsalsolinol or norsalsolinol. Following an initial rapid decline in tyrosine hydroxylation, the residual activity remained stable for 5 h at 37 degrees C. Phosphorylation by protein kinase A facilitates the release of bound catecholamines and is the most prominent mechanism of tyrosine hydroxylase reactivation. Protein kinase A also fully restored enzyme activity after incubation with N-methyl-norsalsolinol, demonstrating that tyrosine hydroxylase inhibition by 6,7-dihydroxylated tetrahydroisoquinolines mimics all essential aspects of catecholamine end-product regulation. Increased levels of N-methyl-norsalsolinol and related tetrahydroisoquinolines are therefore likely to accelerate dopamine depletion in Parkinson's disease.     

Activation of striatal tyrosine hydroxylase by neurocatin,a neuroregulator from mammalian brain

Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of dopamine synthesis in striatal rat synaptosomes. Incubation of intact synaptosomes with neurocatin caused an increase in the rate of dopamine synthesis measured by accumulation of DOPA. The increase is rapid (within two minutes) and dependent on the concentration of added neurocatin. The stimulatory effect of neurocatin on dopamine synthesis occurred only in intact synaptosomes and was almost completely abolished by lysis of the synaptosomes with Triton X-100 or sonification prior to neurocatin addition. The kinetic parameters of tyrosine hydroxylase were measured in lysates prepared from synaptosomes preincubated with neurocatin. These showed that with increasing neurocatin concentration there was an increase in V_max with no significant change in K_M for the pteridine cofactor, compared to control. Activation of tyrosine hydroxylase by neurocatin is at least partially caused by a receptor mediated increase in phosphorylation of the enzyme. Protein kinase C and protein kinase II may be involved in this process.     

Regulation of tyrosine hydroxylase from human pheochromocytoma, bovine adrenal and rat striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum can be activated by Mg²⁺, ATP and cyclic AMP. In pheochromocytoma, this activation is due to a decreased Km for the pterin cofactor, whereas in adrenal medulla, it is a result of an increase in the Vmax. Norepinephrine increases the Km for pterin cofactor for tyrosine hydroxylase from both of these tissues. The Ki for norepinephrine is not altered by the presence of Mg²⁺, ATP and cyclic AMP with enzyme from pheochromocytoma or adrenal medulla. On the other hand, striatal tyrosine hydroxylase shows a two-fold increase in the Ki for dopamine after exposure to Mg²⁺, ATP and cyclic AMP.     

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.     

Regulation of retinal dopamine biosynthesis and tyrosine hydroxylase activity by light

Dopamine (DA)-containing neurons of the rat retina are apparently activated transsynaptically by photic stimulation. Exposure of dark-adapted rats to light increases retinal DA biosynthesis and metabolism. Associated with the light-evoked increase of DA biosynthesis is a rapid activation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis. The activation of TH is characterized by an increased affinity of the enzyme for the pteridine cofactor. Because TH in dark-adapted retinas is apparently not saturated with cofactor, the light-evoked increase of affinity is probably responsible for the observed stimulation of DA biosynthesis. Cyclic AMP (cAMP)-dependent protein phosphorylation in vitro activates TH extracted from dark-adapted retinas, and phosphorylation-induced TH activation is very similar and not additive with light-evoked activation of the enzyme. Incubation of viable cell suspensions of dissociated retinas with 8-bromo cAMP also activates TH, which indicates the availability of sufficient cAMP-dependent protein kinase in the proper subcellular compartment to regulate the enzyme in situ. The DA-containing neurons of the rat retina are tonically inhibited in darkness, and evidence is presented that this tonic inhibition involves direct synaptic input to the DA neurons from gamma-aminobutyric acid-containing amacrine cells. The DA-containing neurons are also subject to feedback inhibition through DA receptors, and to modulation by alpha 2-adrenergic receptors.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Short-term Effects of Endothelins on Tyrosine Hydroxylase Activity and Expression in the Olfactory Bulb of Normotensive Rats

The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca²⁺/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.     

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.     

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.