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.     

Inactivation of Tyrosine Hydroxylase Activity by Ascorbate In Vitro and in Rat PC12 Cells

Abstract: Tyrosine hydroxylase activity is reversibly modulated by the actions of a number of protein kinases and phosphoprotein phosphatases. A previous report from this laboratory showed that low-molecular-weight substances present in striatal extracts lead to an irreversible loss of tyrosine hydroxylase activity under cyclic AMP-dependent phosphorylation conditions. We report here that ascorbate is one agent that inactivates striatal tyrosine hydroxylase activity with an EC₅₀ of 5.9 μM under phosphorylating conditions. Much higher concentrations (100 mM) fail to inactivate the enzyme under nonphosphorylating conditions. Isoascorbate (EC₅₀, 11 μM) and dehydroascorbate (EC₅₀, 970 μM) also inactivated tyrosine hydroxylase under phosphorylating but not under nonphosphorylating conditions. In contrast, ascorbate sulfate was inactive under phosphorylating conditions at concentrations up to 100 mM. Since the reduced compounds generate several reactive species in the presence of oxygen, the possible protecting effects of catalase, peroxidase, and superoxide dismutase were examined. None of these three enzymes, however, afforded any protection against inactivation. We also examined the effects of ascorbate and its congeners on the activity of tyrosine hydroxylase purified to near homogeneity from a rat pheochromocytoma. This purified enzyme was also inactivated by the same agents that inactivated the impure corpus striatal enzyme. Under conditions in which ascorbate almost completely abolished enzyme activity, we found no indication for significant prote-olysis of the purified enzyme as determined by sodium do-decyl sulfate-polyacrylamide gel electrophoresis. We also found that pretreatment of PC12 cells in culture for 4 h with 1 mM ascorbate, dehydroascorbate, or isoascorbate (but not ascorbate sulfate) also decreased tyrosine hydroxylase activity 25–50%. The inactivation seen under in vitro conditions appears to have a counterpart under more physiological conditions.     

Activation of Retinal Tyrosine Hydroxylase In Vitro by Cyclic AMP-Dependent Protein Kinase: Characterization and Comparison to Activation In Vivo by Photic Stimulation

Abstract: Tyrosine hydroxylase in rat retina is activated in vivo as a consequence of photic stimulation. Tyrosine hydroxylase in crude extracts of dark-adapted retinas is activated in vitro by incubation under conditions that stimulate protein phosphorylation by cyclic AMP-dependent protein kinase. Comparison of the activations of the enzyme by photic stimulation in vivo and protein phosphorylation in vitro demonstrated several similarities. Both treatments decreased the apparent K _m of the enzyme for the synthetic pterin cofactor 6MPH₄. Both treatments also produced the same change in the relationships of tyrosine hydroxylase activity to assay pH. When retinal extracts containing tyrosine hydroxylase activated either in vivo by photic stimulation or in vitro by protein phosphorylation were incubated at 25°C, the enzyme was inactivated in a time-dependent manner. The inactivation of the enzyme following both activation in vivo and activation in vitro was partially inhibited by sodium pyrophosphate, an inhibitor of phosphoprotein phosphatase. In addition to these similarities, the activation of tyrosine hydroxylase in vivo by photic stimulation was not additive to the activation in vitro by protein phosphorylation. These data indicate that the mechanism for the activation of tyrosine hydroxylase that occurs as a consequence of light-induced increases of neuronal activity is similar to the mechanism for activation of the enzyme in vitro by protein phosphorylation. This observation suggests that the activation of retinal tyrosine hydroxylase in vivo may be mediated by phosphorylation of tyrosine hydroxylase or some effector molecule associated with the enzyme.     

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.     

Tyrosine Hydroxylase Inactivation Following cAMP-Dependent Phosphorylation Activation

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is activated following phosphorylation by the cAMP-dependent protein kinase (largely by decreasing the K^m of the enzyme for its pterin co-substrate). Following its phosphorylation activation in rat striatal homogenates, we find that tyrosine hydroxylase is inactivated by two distinct processes. Because cAMP is hydrolyzed in crude extracts by a phospho-diesterase, cAMP-dependent protein kinase activity declines following a single addition of cAMP. When tyrosine hydroxylase is activated under these transient phosphorylation conditions, inactivation is accompanied by a reversion of the activated kinetic form (low apparent K^m for pterin co-substrate, ≤0.2 mM) to the kinetic form characteristic of the untreated enzyme (high apparent K^m, ≥1.0 mM). This inactivation is readily reversed by the subsequent addition of cAMP. When striatal tyrosine hydroxylase is activated under constant phosphorylation conditions (incubated with purified cAMP-dependent protein kinase catalytic subunit), however, it is also inactivated. This second inactivation process is irreversible and is characterized kinetically by a decreasing apparent V^max with no change in the low apparent K^m for pterin co-substrate (0.2 mM). The latter inactivation process is greatly attenuated by gel filtration which resolves a low-molecular-weight inactivating factor(s) from the tyrosine hydroxylase. These results are consistent with a regulatory mechanism for tyrosine hydroxylase involving two processes: in the first case, reversible phosphorylaton and dephos-phorylation and, in the second case, an irreversible loss of activity of the phosphorylated form of tyrosine hydroxylase.     

Inactivation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by koningic acid

Koningic acid, a sesquiterpene antibiotic, is a specific inhibitor of the enzyme glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12). In the presence of 3 mM of NAD+, koningic acid irreversibly inactivated the enzyme in a time-dependent manner. The pseudo-first-order rate constant for inactivation (kapp) was dependent on koningic acid concentration in saturate manner, indicating koningic acid and enzyme formed a reversible complex prior to the formation of an inactive, irreversible complex; the inactivation rate (k 3) was 5.5.10(-2) s-1, with a dissociation constant for inactivation (Kinact) of 1.6 microM. The inhibition was competitive against glyceraldehyde 3-phosphate with a Ki of 1.1 microM, where the Km for glyceraldehyde 3-phosphate was 90 microM. Koningic acid inhibition was uncompetitive with respect to NAD+. The presence of NAD+ accelerated the inactivation. In its absence, the charcoal-treated NAD+-free enzyme showed a 220-fold decrease in apparent rate constant for inactivation, indicating that koningic acid sequentially binds to the enzyme next to NAD+. The enzyme, a tetramer, was inactivated when maximum two sulfhydryl groups, possibly cysteine residues at the active sites of the enzyme, were modified by the binding of koningic acid. These observations demonstrate that koningic acid is an active-site-directed inhibitor which reacts predominantly with the NAD+-enzyme complex.     

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.     

The rapid activation of tyrosine hydroxylase by the subcutaneous injection of formaldehyde

Ten minutes following the subcutaneous injection of formaldehyde, there is a 2-fold activation of adrenal medulla tyrosine hydroxylase. Enzyme activation appears to involve a shift of a fraction of the low affinity form of the enzyme to a higher affinity form. In the nonstessed rat approximately 29% of adrenal TH is in the activated (low Km) form. Following formaldehyde injection, approximately 54% of the enzyme is in the activated (low Km) form. The addition of cyclic AMP, ATP and Mg⁺⁺, in the presence of endogenous cyclic AMP-dependent protein kinase, to the enzyme obtained from adrenals of either the stressed or nonstressed rats increased the activity of both enzyme preparations to the same level. These data indicate that acute stress activates adrenal tyrosine hydroxylase in the rat and that a cyclic AMP-dependent protein phosphorylating system mediates the activation.     

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.     

Properties of purified L-ornithine decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO2.h-1.mg-1. The purified enzyme was rather stable at 37 degrees C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the Km for the substrate L-ornithine was 0.11 mM, and the Km for the cofactor pyridoxal 5-phosphate was 0.12 microM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated Ki of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a Ki of 1.6 mM and by alpha-difluoromethylornithine with a Ki of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Splicing Pattern of Gsα mRNA in Human and Rat Brain

The enzyme tyrosine hydroxylase catalyzes the first step in the biosynthesis of dopamine, norepinephrine, and epinephrine. Tyrosine hydroxylase is a substrate for cyclic AMP-dependent protein kinase as well as other protein kinases. We determined the Km and Vmax of rat pheochromocytoma tyrosine hydroxylase for cyclic AMP-dependent protein kinase and obtained values of 136 microM and 7.1 mumol/min/mg of catalytic subunit, respectively. These values were not appreciably affected by the substrates for tyrosine hydroxylase (tyrosine and tetrahydrobiopterin) or by feedback inhibitors (dopamine and norepinephrine). The high Km of tyrosine hydroxylase correlates with the high content of tyrosine hydroxylase in catecholaminergic cells. We also determined the kinetic constants for peptides modeled after actual or potential tyrosine hydroxylase phosphorylation sites. We found that the best substrates for cyclic AMP-dependent protein kinase were those peptides corresponding to serine 40. Tyrosine hydroxylase (36-46), for example, exhibited a Km of 108 microM and a Vmax of 6.93 mumol/min/mg of catalytic subunit. The next best substrate was the peptide corresponding to serine 153. The peptide containing the sequence conforming to serine 19 was a very poor substrate, and that conforming to serine 172 was not phosphorylated to any significant extent. The primary structure of the actual or potential phosphorylation sites is sufficient to explain the substrate behavior of the native enzyme.     

Cell-free detection and characterization of a novel nerve growth factor-activated protein kinase in PC12 cells

We have developed a cell-free assay to detect and characterize nerve growth factor (NGF)-activated protein kinase activity. Cultured PC12 cells were briefly exposed to NGF, and extracts of these were assayed for phosphorylating activity using exogenously added tyrosine hydroxylase as substrate. Tyrosine hydroxylase was employed since it is an endogenous substrate of NGF-regulated kinase activity and is activated by phosphorylation. In the cell-free assay, extracts prepared from NGF-treated cells yielded a 2-3-fold greater incorporation of phosphate into tyrosine hydroxylase as compared with extracts of control, NGF-untreated cells. Activation did not occur, however, if NGF was added directly to cell extracts. The NGF-stimulated phosphorylating activity appeared to be due to regulation of a protein kinase rather than of a phosphoprotein phosphatase. Characterization of the kinase (designated as kinase N) showed that it is soluble, is detectably activated within 1-3 min after cells are exposed to NGF and maximally activated by 10 min, is half-maximally activated with 0.5 nM NGF and maximally activated with 1 nM NGF, is detectable in the presence of either Mg2+ or Mn2+ but does not require Ca2+, does not require nonmacromolecular cofactors, can use histone H1 as a substrate, and exhibits a 2-fold increase in apparent Vmax in response to NGF but does not undergo a significant change in apparent Km for either ATP or GTP. A number of characteristics of kinase N were assessed including susceptibility to inhibitors, substrate specificity, cofactor requirements, ATP dependence, and lack of down-regulation by prolonged expose to a phorbol ester. These studies indicated that it lacks tyrosine kinase activity and is distinct from a variety of well-characterized protein kinases including cAMP-dependent protein kinase, protein kinase C (Ca2+/phospholipid-dependent enzyme), Ca2+/calmodulin-dependent kinase, and casein kinase II. Preliminary purification data show that the kinase has a basic pI and that it has an apparent Mr of 22,000-25,000. The only amino acid in tyrosine hydroxylase found to be phosphorylated by the semipurified kinase is serine.     

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.     

Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust

The metabolic transformation of tyrosine (TYR) by the decarboxylase and hydroxylase enzymes was investigated in the central nervous system of the locust, Locusta migratoria. It has been demonstrated that the key amino acids, 3,4-dihydroxyphenylalanine (DOPA), 5-hydroxytryptophan (5HTP) and tyrosine are decarboxylated in all part of central nervous system. DOPA and 5HTP decarboxylase activities show parallel changes in the different ganglia, but the rank order of the activity of TYR decarboxylase is different. Enzyme purification has revealed that the molecular weights of TYR decarboxylase and DOPA/5HTP decarboxylase are 370,000 and 112,000, respectively. The decarboxylation of DOPA by DOPA/5HTP decarboxylase is stimulated, whereas the decarboxylation of DOPA by TYR decarboxylase is inhibited in the presence of the cofactor pyridoxal-5'-phosphate. TYR hydroxylase could not be detected and 3H-TYR is found to be metabolised to tyramine (TA), but not to DOPA. The haemolymph contains a significant concentration of DOPA (120 pmol/100 microl haemolymph), and the ganglia incorporates DOPA from the haemolymph by a high affinity uptake process (K(M)=12 microM and V(max)=24 pmol per ganglion/10 min). Our results suggest that no tyrosine hydroxylase is present in the locust CNS and the DOPA uptake into the ganglia by a high affinity uptake process as well as the DOPA decarboxylase enzyme may be responsible for the regulation of the ganglionic dopamine (DA) level. Two types of decarboxylases exist, one of them decarboxylating DOPA and 5HTP (DOPA/5HTP decarboxylase), other decarboxylating TYR (TYR decarboxylase). The DOPA/5HTP decarboxylase enzyme present in the insect brain may correspond to the 5HTP/DOPA decarboxylase in vertebrate brain, whereas TYR decarboxylase is characteristic only for the insect brain.     

Effects of phosphorylation by protein kinase A on binding of catecholamines to the human tyrosine hydroxylase isoforms

Tyrosine hydroxylase (TyrH), the catalyst for the key regulatory step in catecholamine biosynthesis, is phosphorylated by cAMP-dependent protein kinase A (PKA) on a serine residue in a regulatory domain. In the case of the rat enzyme, phosphorylation of Ser40 by PKA is critical in regulating the enzyme activity; the effect of phosphorylation is to relieve the enzyme from inhibition by dopamine and dihydroxyphenylalanine (DOPA). There are four isoforms of human tyrosine hydroxylase (hTyrH), differing in the size of an insertion after Met30. The effects of phosphorylation by PKA on the binding of DOPA and dopamine have now been determined for all four human isoforms. There is an increase of about two-fold in the Kd value for DOPA for isoform 1 upon phosphorylation, from 4.4 to 7.4 microM; this effect decreases with the larger isoforms such that there is no effect of phosphorylation on the Kd value for isoform 4. Dopamine binds more much tightly, with Kd values less than 3 nM for all four unphosphorylated isoforms. Phosphorylation decreases the affinity for dopamine at least two orders of magnitude, resulting in Kd values of about 0.1 microM for the phosphorylated human enzymes, due primarily to increases in the rate constant for dissociation of dopamine. Dopamine binds about two-fold less tightly to the phosphorylated isoform 1 than to the other three isoforms. The results extend the regulatory model developed for the rat enzyme, in which the activity is regulated by the opposing effects of catecholamine binding and phosphorylation by PKA. The small effects on the relatively high Kd values for DOPA suggest that DOPA levels do not regulate the activity of hTyrH.     

Tyrosine hydroxylase. Activation by protein phosphorylation and end product inhibition.

Protein phosphorylation activates tyrosine hydroxylase in crude extracts of rat striatum, hypothalamus, and adrenal glands by a reduction in the apparent Km value for 6-methyltetrahydropterin. Removal of endogenous catecholamines by gel filtration or cation exchange results in a similar activation. Phosphorylation causes only a small additional reduction in the apparent Km for reduced pterin in striatal extracts from which catecholamines have been removed. Kinetic analysis indicates that protein phosphorylation causes a significant increase in the Ki for end product dopamine, whereas gel filtration or cation exchange treatment has little effect on the dopamine Ki value. None of the above treatments appears to change the molecular weight of the enzyme. At physiological concentrations of dopamine, the increase in Ki by phosphorylation would effectively release tyrosine hydroxylase from end product feedback inhibition.     

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.     

Regulation of tyrosine hydroxylase activity in the rat hypothalamus with gamma-aminobutyric acid

It is established that GABA interacts with tyrosine hydroxylase through the allosteric site which is not identical to sites of tyrosine, DOPA, pterin cofactor, dopamine binding. This interaction is very significant in the GABA influence on the regulation of the tyrosine hydroxylase activity by presynaptic receptors. GABA is supposed to be able to cause dissociation of oligomeric forms of tyrosine hydroxylase.     

Effect of spermine on tyrosine hydroxylase activity before and after phosphorylation by cyclic AMP-dependent protein kinase

The effect of spermine on tyrosine hydroxylase (TH) activity purified from bovine adrenal medulla was examined before and after phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase (A-kinase). Before phosphorylation, spermine (less than 1 mM) inhibited the enzymatic activity, and negative cooperative effect of spermine on TH (Hill coefficient = 0.7) was observed from the kinetic analysis concerning 6-methyl-5,6,7,8-tetrahydropterin (6MPH4). Spermine interacted noncompetitively toward tyrosine and the Ki for spermine was calculated to be 68 microM. Phosphorylation abolished the ability of spermine to inhibit TH activity in a negative cooperative manner against the pterin cofactor, and also increased four-fold the Ki value against the substrate. These results suggest that spermine may inhibit TH activity by interacting with the pterin binding site of the enzyme molecule in a manner of negative cooperativity, and that this inhibition is reversed by the conformational change of regulatory domain of TH after phosphorylation by A-kinase.