Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Role of C6 chirality of tetrahydropterin cofactor in catalysis and regulation of tyrosine and phenylalanine hydroxylases. 168 99

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.
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PMID:Interaction of tyrosine hydroxylase with ribonucleic acid and purification with DNA-cellulose or poly(A)-sepharose affinity chromatography. 244 76

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.
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PMID:Changes in the cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues. 256 33

Tyrosine hydroxylase phosphatase activity in rat caudate nucleus was separated into three peaks by chromatography on DEAE-cellulose. [32P]Tyrosine hydroxylase phosphorylated by cyclic AMP-dependent protein kinase was dephosphorylated only by the major peak eluting at 0.3 M NaCl, while tyrosine hydroxylase phosphorylated by Ca2+-calmodulin-dependent protein kinase was also dephosphorylated by two calcium-inhibited phosphatases. The Vmax of the enzyme in the major DEAE peak was increased by 10 microM tetrahydrobiopterin (BH4) from 0.78 to 5.0 fmol min-1 mg-1 while the Km was only slightly affected, increasing from 45 to 62 pM. The activation could not be reversed by dilution. On Sephadex G-200, the enzyme was found to consist of two major forms with molecular masses of 420 and 100 kDa. In contrast to the activation of liver phosphatases by freezing with beta-mercaptoethanol, activation by tetrahydrobiopterin was not associated with a shift in the molecular weight of the phosphatase to lower molecular weight forms. Other reduced pterins, including tetrahydroneopterin, 6-methyltetrahydropterin, and 5-methyltetrahydrofolate, also activated the enzyme, while oxidized pterins had no effect. GTP, the metabolic precursor of tetrahydrobiopterin, was a potent inhibitor of the phosphatase reaction, inhibiting by 65% at a concentration of 1 microM. These findings suggest a close regulatory interrelationship between the tetrahydrobiopterin synthetic pathway and catecholamine biosynthesis.
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PMID:Activation of rat caudate tyrosine hydroxylase phosphatase by tetrahydropterins. 289 Jun 38

Y-1 adrenal cortical tumor cells in culture, which contain substantial amounts of tetrahydrobiopterin [6R-(L-erythro-1',2'-dihydroxypropyl)5,6,7,8-tetrahydropterin] (BH4) and GTP cyclohydrolase (GTP-CH), were used to study the regulation of BH4 biosynthesis by ACTH and cAMP. ACTH produced a dose-dependent increase in steroidogenesis, BH4 levels and GTP-CH activity. Maximal stimulation of BH4 biosynthesis occurred at the same concentration of ACTH that caused maximal stimulation of steroidogenesis. ACTH-(1-24) was more potent than ACTH-(1-39). The stimulation of BH4 biosynthesis by ACTH was dependent on cell density, being greater at lower cell densities, but was independent of time in culture. The lack of stimulation by ACTH at higher cell densities was due to an increase in the specific activity of GTP-CH in the control cells as density increased. This increase may be due in part to the increased release of steroids, since exogenous steroids added to low density cultures also resulted in an increase in the specific activity of the enzyme. Addition of steroids had no effect on ACTH-dependent stimulation of BH4 biosynthesis at low cell densities. (Bu)2cAMP, 8-bromo-cAMP, and forskolin all produced time- and dose-dependent increases in BH4 levels, GTP-CH activity, and steroidogenesis. Maximum increases in GTP-CH and BH4 occurred at concentrations similar to those required for maximal stimulation of steroidogenesis. In the Kin-8 mutant of Y-1 cells, which has a type 1 cAMP-dependent protein kinase with an altered regulatory subunit, ACTH was unable to increase BH4 levels or GTP-CH activity at a concentration that produced maximal stimulation of BH4 and steroid biosynthesis in the parent Y-1 line. These studies indicate that Y-1 cells in culture are useful for studying the regulation of BH4 biosynthesis in the adrenal cortex.
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PMID:Regulation of tetrahydrobiopterin biosynthesis in cultured adrenal cortical tumor cells by adrenocorticotropin and adenosine 3',5'-cyclic monophosphate. 300 41

Rat liver phenylalanine hydroxylase catalyzes the tetrahydropterin-dependent oxidation of phenylalanine to tyrosine, according to equation 1. In addition to the naturally-occurring coenzyme, tetrahydrobiopterin (BH4), certain synthetic analogs of BH4 such as 6-methyltetrahydropterin (6MPH4) have high cofactor activity. (formula; see text) The hydroxylase can be activated by a variety of reversible and irreversible modifications, including those caused by partial proteolysis, by interaction with phospholipids such as lysolecithin, by alkylation of a single sulfhydryl group, by phosphorylation catalyzed by cAMP-dependent protein kinase, and by preincubation with its substrate, phenylalanine. All of these modes of activation greatly increase the hydroxylase activity in the presence of BH4, whereas the activity in the presence of 6MPH4 is increased only slightly. The ratio of hydroxylase activity in the presence of BH4 compared to the activity in the presence of 6MPH4, therefore, is a useful index of the state of activation of the enzyme. Of the various activation mechanisms listed above, only phosphorylation of the enzyme and phenylalanine-activation appear to operate in vivo. The evidence indicates that these two regulatory mechanisms act synergistically. Thus, phosphorylation of the enzyme by cAMP-dependent protein kinase is stimulated by phenylalanine, especially in the presence of BH4, (which by itself inhibits), whereas phosphorylation sensitizes the enzyme to activation by phenylalanine. One of the consequences of these interlocking control mechanisms is to enhance the responsiveness of the activity of the hydroxylase to alterations in tissue levels of phenylalanine. As a result, elevated concentrations of phenylalanine can be rapidly metabolized, thereby protecting the fetal and neonatal brain from possible damage by excess phenylalanine.
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PMID:Regulation of the activity of hepatic phenylalanine hydroxylase. 302 51

The rate of phosphorylation of phenylalanine 4-monooxygenase by the cAMP-dependent protein kinase was found to be under substrate-directed regulation. Thus, L-phenylalanine made the hydroxylase a better substrate for the kinase, whereas the cofactor l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) was a negative effector. The dephosphorylation of the enzyme by the kinase in the presence of high concentrations of MgADP was also stimulated by phenylalanine and inhibited by BH4. A kinetic analysis indicated that the effects of phenylalanine and BH4 were mediated by distinct sites coupled by a free energy of 3.2 kJ X mol-1. Among the ligands tested, only phenylalanine and BH4 affected the phosphorylation of the hydroxylase at physiologically relevant concentrations. Whereas higher concentrations of several naturally occurring or synthetic amino acids acted like phenylalanine, the widely used synthetic cofactor 6,7-dimethyltetrahydropterin did not mimic the effect of BH4. Less phenylalanine was required to activate the phosphorylated hydroxylase (0.9 mol of phosphate/subunit) than the dephosphorylated enzyme (0.07 mol of phosphate/subunit). This was true whether BH4 was present or not. In conclusion, the substrate phenylalanine makes the hydroxylase more prone to cAMP-dependent phosphorylation, which in turn sensitizes the enzyme towards allosteric activation by phenylalanine. The joint operation of these mechanisms in vivo would increase the efficiency with which phenylalanine controls the activity of the enzyme.
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PMID:The effect of ligands of phenylalanine 4-monooxygenase on the cAMP-dependent phosphorylation of the enzyme. 647 1

Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C (Ca2+/phospholipid-dependent protein kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 +/- 0.05 (n = 4) and 0.74 +/- 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent protein kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent protein kinase II. Phosphorylation of sepiapterin reductase by these protein kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca(2+)-activated protein kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
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PMID:Phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin. 813 44

Unipolar depression, alcoholism and suicide have become more common over the past decades. Genetic studies have attempted to link (bipolar) affective disorder to the short arm of chromosome 11 (where the loci for insulin, insulin growth factor (IGF), tyrosine hydroxylase (TH) and h-ras-oncogene are located) but these have failed. Since TH and the insulin receptor require phosphorylation by protein kinases, then a defect of the h-ras-oncogene or its products (p21) could disorder both these systems and compromise catecholaminergic transmission in neurones and energy flow in glial cells. This could lead not only to a predisposition to depression ('trait markers') but to neurotoxic damage, predisposed by inadequate cytosol Mg2+ levels of hypometabolism. Tyrosine, tryptophan and phenylalanine hydroxylases all require tetrahydrobiopterin (BH4) which allosterically regulates its own activity as well as that of these enzymes. Anything which impairs this cofactor could lead to overt depression in predisposed individuals, and the heterocyclic amines are being increasingly implicated. These substances are derived from fried and broiled meats, azo food dyes, soft drinks and hard candies, but particularly from cigarette and petroleum fumes. The heterocyclic amines can inhibit aromatic-l-amino-acid-decarboxylase (AADC) as well as the hydroxylases reversibly, but BH4 is inhibited noncompetitively. Thus, susceptible individuals (those with inherited defective protein kinase phosphorylation) might be 'tipped over' by chronic exposure to these neurotoxins. The rising incidence of unipolar depression-associated morbidity could be significantly linked to increasing levels of heterocyclic amines in the developed nations.
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PMID:The 'cerebral diabetes' paradigm for unipolar depression. 814 51

Recombinant human liver phenylalanine hydroxylase (PAH) expressed in Escherichia coli has been purified to homogeneity. The recombinant enzyme exists in solution as a mixture of 80% tetramers and 20% dimers. A study of the kinetic properties of the enzyme indicates that compared to the recombinant and the native rat liver enzymes, the recombinant human enzyme is in an activated state. This conclusion is supported by the finding that its catalytic activity is only marginally stimulated by incubation with either phenylalanine or lysolecithin. In contrast, the native and the recombinant rat liver enzymes are activated 8- to 25-fold, respectively, when preincubated with phenylalanine or lysolecithin. In the absence of activators, the ratio of the hydroxylase activity in the presence of 6-methyl-5,6,7,8-tetrahydropterin compared to the activity in the presence of (6R)-5,6,7,8-tetrahydrobiopterin (BH4), which is an index of the state of activation of the enzyme, is 4 for the human recombinant PAH compared to a value of 12 for the recombinant rat liver enzyme. Furthermore, the Km for phenylalanine in the presence of BH4 is 0.050 mM, a value that is one-fifth that of the recombinant rat liver enzyme. Covalent modification of the human enzyme by phosphorylation with protein kinase A provides further evidence that the human enzyme is in a substantially activated state. Phosphorylation, which results in the incorporation of 0.6 mol of phosphate/mol of subunit, leads to only a modest activation of 1.5-fold compared to about a 3-fold activation seen after phosphorylation of the native and the recombinant rat liver enzymes. Moreover, the recombinant human liver enzyme is less sensitive than the rat liver enzyme to stimulation by lysolecithin when tryptophan is the substrate. Just as is true for the rat liver enzyme, the apparent Km values for tryptophan and pheylalanine vary with the pterin cofactor employed. The ability of 7-tetrahydrobiopterin (7-BH4) to substitute for the natural cofactor tetrahydrobiopterin has been studied in vitro. The apparent Km for 7-BH4 for the recombinant human enzyme is 0.2 mM and the Km for phenylalanine is 0.05 mM. The hydroxylase reaction is severely inhibited by 7-BH4 in the presence of physiological concentrations of BH4. This inhibition can be overcome by a decrease in the concentration of phenylalanine. The implications of these novel properties of human PAH for phenylalanine homoestasis in man are discussed.
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PMID:Recombinant human phenylalanine hydroxylase: novel regulatory and structural properties. 880 57


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