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Phillip W. Dickson - One of the best experts on this subject based on the ideXlab platform.
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Tyrosine Hydroxylase phosphorylation: regulation and consequences.
Journal of neurochemistry, 2004Co-Authors: Peter R. Dunkley, Larisa Bobrovskaya, Mark E. Graham, Ellak I. Von Nagy-felsobuki, Phillip W. DicksonAbstract:The rate-limiting enzyme in catecholamine synthesis is Tyrosine Hydroxylase. It is phosphorylated at serine (Ser) residues Ser8, Ser19, Ser31 and Ser40 in vitro, in situ and in vivo. A range of protein kinases and protein phosphatases are able to phosphorylate or dephosphorylate these sites in vitro. Some of these enzymes are able to regulate Tyrosine Hydroxylase phosphorylation in situ and in vivo but the identity of the kinases and phosphatases is incomplete, especially for physiologically relevant stimuli. The stoichiometry of Tyrosine Hydroxylase phosphorylation in situ and in vivo is low. The phosphorylation of Tyrosine Hydroxylase at Ser40 increases the enzyme's activity in vitro, in situ and in vivo. Phosphorylation at Ser31 also increases the activity but to a much lesser extent than for Ser40 phosphorylation. The phosphorylation of Tyrosine Hydroxylase at Ser19 or Ser8 has no direct effect on Tyrosine Hydroxylase activity. Hierarchical phosphorylation of Tyrosine Hydroxylase occurs both in vitro and in situ, whereby the phosphorylation at Ser19 increases the rate of Ser40 phosphorylation leading to an increase in enzyme activity. Hierarchical phosphorylation depends on the state of the substrate providing a novel form of control of Tyrosine Hydroxylase activation.
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Identification of Gln313 and Pro327 as Residues Critical for Substrate Inhibition in Tyrosine Hydroxylase
Journal of neurochemistry, 2002Co-Authors: Noelene Sheila Quinsey, Catherine M. Lenaghan, Phillip W. DicksonAbstract:Rat Tyrosine Hydroxylase was expressed in Escherichia coli. High-level expression was obtained after incubation at 27°C for 18 h. The smallest fragment of Tyrosine Hydroxylase that gave a soluble active molecule was from Leu 188 to Phe 456 . This fragment corresponds directly to the section of phenylalanine Hydroxylase that had previously been shown to be this enzyme's catalytic core region. It has been shown that Glu 286 plays a critical role in pterin function in phenylalanine Hydroxylase. The corresponding residue in Tyrosine Hydroxylase (Glu 332 ) has no significant role in pterin function. Substitution of a leucine for a proline at position 327 in Tyrosine Hydroxylase produces a molecule with a K m for tetrahydrobiopterin 20-fold higher than that of the wild-type molecule, whereas the same substitution at the corresponding residue in phenylalanine Hydroxylase (Pro 281 ) has no effect on the kinetic constant for the cofactor. This suggests that corresponding residues in phenylalanine Hydroxylase and Tyrosine Hydroxylase can have different roles in pterin function. Substitution of a leucine for a proline at position 281 in phenylalanine Hydroxylase increases the K m for phenylalanine >20-fold over that of the wild-type. Substitution of leucine or alanine for Pro 327 or a glutamic acid for Gln 313 in Tyrosine Hydroxylase eliminates the substrate inhibition shown by wild-type Tyrosine Hydroxylase.
Paul F. Fitzpatrick - One of the best experts on this subject based on the ideXlab platform.
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Specificity of the MAP kinase ERK2 for phosphorylation of Tyrosine Hydroxylase.
Archives of biochemistry and biophysics, 2004Co-Authors: Montserrat Royo, S. Colette Daubner, Paul F. FitzpatrickAbstract:Abstract Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of Tyrosine Hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of Tyrosine Hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2 mol of phosphate into each subunit of Tyrosine Hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A Tyrosine Hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.
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Reversing the Substrate Specificities of Phenylalanine and Tyrosine Hydroxylase: Aspartate 425 of Tyrosine Hydroxylase Is Essential for l-DOPA Formation†
Biochemistry, 2000Co-Authors: S. C. Daubner, J. Melendez, Paul F. FitzpatrickAbstract:The catalytic domains of the pterin-dependent enzymes phenylalanine Hydroxylase and Tyrosine Hydroxylase are homologous, yet differ in their substrate specificities. To probe the structural basis for the differences in specificity, seven residues in the active site of phenylalanine Hydroxylase whose side chains are dissimilar in the two enzymes were mutated to the corresponding residues in Tyrosine Hydroxylase. Analysis of the effects of the mutations on the isolated catalytic domain of phenylalanine Hydroxylase identified three residues that contribute to the ability to hydroxylate Tyrosine, His264, Tyr277, and Val379. These mutations were incorporated into full-length phenylalanine Hydroxylase and the complementary mutations into Tyrosine Hydroxylase. The steady-state kinetic parameters of the mutated enzymes showed that the identity of the residue in Tyrosine Hydroxylase at the position corresponding to position 379 of phenylalanine Hydroxylase is critical for dihydroxyphenylalanine formation. The rela...
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Mechanistic Studies of Tyrosine Hydroxylase
Advances in experimental medicine and biology, 1993Co-Authors: Paul F. FitzpatrickAbstract:Tyrosine Hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamine neurotransmitters, the hydroxylation of Tyrosine to dihydroxyphenylalanine1. The other substrates for the reaction are molecular oxygen and a tetrahydropterin. In addition, Tyrosine Hydroxylase requires one atom of ferrous iron per active site for activity2,3; the role of the iron atom is unknown. While the central position of Tyrosine Hydroxylase in the function of the central nervous system has resulted in a great deal of interest in the enzyme over the years, very little is known about the actual mechanism of catalysis. This report describes recent studies towards elucidating the mechanism of this important monooxygenase.
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The amino acid substrate of bovine Tyrosine Hydroxylase.
Neurochemistry international, 1992Co-Authors: Marc M. Meyer, Paul F. FitzpatrickAbstract:Abstract Tyrosine Hydroxylase catalyzes the tetrahydropterin-dependent hydroxylation of Tyrosine to form 3,4-dihydroxyphenylalanine. Several nonphysiological aromatic amino acids have been examined as inhibitors and substrates for bovine adrenal Tyrosine Hydroxylase. The Ki values for para-substituted phenylalanines increase as the size of the substituent increases. For each A2 increase in surface area of the substituent, the free energy of binding becomes 50 cal more positive. Replacement of the phenyl ring with a pyridyl ring decreases the affinity about one order of magnitude. A number of these aromatic amino acids are also substrates for the enzyme. The KM values again increase in size with increasing size of the substituent, but the Vmax value is independent of the reactivity of the amino acid. The effect of size on binding is consistent with a tight interaction between the para position region of the substrate and the enzyme. The lack of a change in the Vmax value is consistent with the rate-limiting step in catalysis by bovine Tyrosine Hydroxylase being formation of the hydroxylating intermediate rather than hydroxylation of the amino acid. These results will be useful in designing mechanism-based inhibitors of catecholamine biosynthesis and establish that the mechanisms of rat and bovine Tyrosine Hydroxylase do not differ significantly.
John W. Haycock - One of the best experts on this subject based on the ideXlab platform.
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Elevated Tyrosine Hydroxylase in the Locus Coeruleus of Suicide Victims
Journal of neurochemistry, 2002Co-Authors: Gregory A. Ordway, Karen Smith, John W. HaycockAbstract:: The amounts of Tyrosine Hydroxylase protein in locus coeruleus from nine pairs of antidepressant-free suicide victims and age-matched, sudden-death control cases were determined by quantitative blot immunolabeling of cryostat-cut sections from the caudal portion of the nucleus. In each of the nine age-matched pairs, the concentration of Tyrosine Hydroxylase was greater in the sample from the suicide victim, with values ranging from 108 to 172% of the matched control value (\-x = 136%). By contrast, there were no differences in the concentrations of neuron-specific enolase protein in the same set of samples. Similarly, the number of neuromelanin-containing cells, counted in sections of locus coeruleus adjacent to those taken for blot immunolabeling analyses, did not differ between the two groups. These data indicate that locus coeruleus neurons from suicide victims contain higher than normal concentrations of Tyrosine Hydroxylase, thus raising the possibility that the expression of Tyrosine Hydroxylase in locus coeruleus may be relevant in the pathophysiology of suicide.
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Four isoforms of Tyrosine Hydroxylase are expressed in human brain.
Neuroscience, 1993Co-Authors: David A. Lewis, Darlene S. Melchitzky, John W. HaycockAbstract:In contrast to nonprimate species, the RNA for human Tyrosine Hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human Tyrosine Hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human Tyrosine Hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human Tyrosine Hydroxylase protein forms to determine the presence and distribution of each human Tyrosine Hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human Tyrosine Hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human Tyrosine Hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human Tyrosine Hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human Tyrosine Hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human Tyrosine Hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.
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Vasoactive intestinal polypeptide induces Tyrosine Hydroxylase in PC12 cells.
The Journal of biological chemistry, 1991Co-Authors: Margaret Wessels-reiker, John W. Haycock, Allyn C. Howlett, Randy StrongAbstract:Physiological stress induces Tyrosine Hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, via trans-synaptic mechanisms within the adrenal medulla. Previous studies have implicated cAMP as a second messenger capable of inducing Tyrosine Hydroxylase; however, it is unclear whether any receptor coupled to adenylate cyclase mediates Tyrosine Hydroxylase induction. Recently, vasoactive intestinal polypeptide, whose receptor is coupled to adenylate cyclase in many tissues, has been shown to meet many of the criteria for a neuromodulator within the adrenal medulla. We therefore undertook a series of studies to determine whether vasoactive intestinal polypeptide may induce Tyrosine Hydroxylase in PC12 cells, a cell line derived from rat adrenal medulla. Here we report that vasoactive intestinal polypeptide produces a transient, time- and concentration-dependent increase in Tyrosine Hydroxylase mRNA levels which is followed by a stable increase in Tyrosine Hydroxylase protein. The increase in Tyrosine Hydroxylase mRNA does not occur in a mutant PC12 cell line deficient in cAMP-dependent protein kinase activity, indicating that the effect of vasoactive intestinal polypeptide is mediated through the cAMP second messenger pathway. This is the first report demonstrating that a neuromodulator which acts on an adenylate cyclase-coupled receptor can induce Tyrosine Hydroxylase.
A W Tank - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Tyrosine Hydroxylase gene transcription rate and Tyrosine Hydroxylase mRNA stability by cyclic AMP and glucocorticoid.
Molecular pharmacology, 1992Co-Authors: L H Fossom, Carol R. Sterling, A W TankAbstract:Tyrosine Hydroxylase mRNA is induced in rat pheochromocytoma PC18 cells by cAMP analogs and glucocorticoids. Previous studies have shown that these increases in Tyrosine Hydroxylase mRNA are due at least in part to stimulation of the Tyrosine Hydroxylase gene. However, the involvement of post-transcriptional mechanisms in the regulation of Tyrosine Hydroxylase mRNA by these inducing agents has not been investigated. In the present study, using nuclear run-on assays we show that the relative transcription rate of the Tyrosine Hydroxylase gene is stimulated 2-5-fold within 20 min after treatment of PC18 cells with cAMP analogs or dexamethasone and that the rate of transcription remains elevated 2-3-fold for at least 24 hr in the continual presence of these inducing agents. Pulse-labeling experiments using 4-thiouridine indicate that the rate of synthesis of Tyrosine Hydroxylase mRNA is increased approximately 3-fold or 10-fold after treatment with either a cyclic AMP analog or dexamethasone, respectively. These increases in rates of synthesis agree well with the fold increases in Tyrosine Hydroxylase mRNA levels after treatment with these inducers. Treatment of the cells with cycloheximide lowers the basal relative transcription rate of the Tyrosine Hydroxylase gene 2-3-fold; however, the relative transcription rate of the Tyrosine Hydroxylase gene is still elevated in cells treated with either dexamethasone or cAMP analogs in the presence of cycloheximide, compared with the transcription rate of the gene in cells treated with cycloheximide alone. These results indicate that protein synthesis is not required for the short term regulation of the gene by these inducing agents. The apparent t1/2 for Tyrosine Hydroxylase mRNA has been estimated by two different procedures, approach to steady state kinetics and pulse-chase analysis. Both procedures yield an estimated apparent t1/2 of approximately 6-9 hr for Tyrosine Hydroxylase mRNA under basal culture conditions. Dexamethasone does not substantially alter this apparent t1/2 value; however, cAMP appears to lower this apparent t1/2 value transiently. Our results suggest that cAMP and glucocorticoid regulate Tyrosine Hydroxylase mRNA levels primarily by stimulating the transcription rate of the Tyrosine Hydroxylase gene; however, cAMP may also regulate the stability of the mRNA for a short period of time, such that it is induced more rapidly in the cells.
Kent E. Vrana - One of the best experts on this subject based on the ideXlab platform.
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Complex molecular regulation of Tyrosine Hydroxylase
Journal of Neural Transmission, 2014Co-Authors: Izel Tekin, Robert Roskoski, Nurgul Carkaci-salli, Kent E. VranaAbstract:Tyrosine Hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is strictly controlled by several interrelated regulatory mechanisms. Enzyme synthesis is controlled by epigenetic factors, transcription factors, and mRNA levels. Enzyme activity is regulated by end-product feedback inhibition. Phosphorylation of the enzyme is catalyzed by several protein kinases and dephosphorylation is mediated by two protein phosphatases that establish a sensitive process for regulating enzyme activity on a minute-to-minute basis. Interactions between Tyrosine Hydroxylase and other proteins introduce additional layers to the already tightly controlled production of catecholamines. Tyrosine Hydroxylase degradation by the ubiquitin–proteasome coupled pathway represents yet another mechanism of regulation. Here, we revisit the myriad mechanisms that regulate Tyrosine Hydroxylase expression and activity and highlight their physiological importance in the control of catecholamine biosynthesis.
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Intricate Regulation of Tyrosine Hydroxylase Activity and Gene Expression
Journal of neurochemistry, 2002Co-Authors: Sean C. Kumer, Kent E. VranaAbstract:Tyrosine Hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of Tyrosine Hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby Tyrosine Hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of Tyrosine Hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady-state levels of active Tyrosine Hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation-dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate Tyrosine Hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.
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Dopamine, in the presence of tyrosinase, covalently modifies and inactivates Tyrosine Hydroxylase
Journal of neuroscience research, 1998Co-Authors: Alan H. Stokes, Robert Roskoski, Kent E. VranaAbstract:Dopamine has been implicated as a potential mediating factor in a variety of neurodegenerative disorders. Dopamine can be oxidized to form a reactive dopamine quinone that can covalently modify cellular macromolecules including protein and DNA. This oxidation can be enhanced through various enzymes including tyrosinase and/or prostaglandin H synthase. One of the potential targets in brain for dopamine quinone damage is Tyrosine Hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis. The present studies demonstrated that dopamine quinone, the formation of which was enhanced through the activity of the melanin biosynthetic enzyme, tyrosinase, covalently modified and inactivated Tyrosine Hydroxylase. Dihydroxyphenylalanine (DOPA; the catechol-containing precursor of dopamine) also inactivated Tyrosine Hydroxylase under these conditions. Catecholamine-mediated inactivation occurred with both purified Tyrosine Hydroxylase as well as enzyme present in crude pheochromocytoma homogenates. Inactivation was associated with covalent incorporation of radiolabelled dopamine into the enzyme as assessed by immunoprecipitation, size exclusion chromatography, and denaturing sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. Furthermore, the covalent modification and inactivation of Tyrosine Hydroxylase was blocked by antioxidant compounds (dithiothreitol, reduced glutathione, or NADH). In addition to kinetic feedback inhibition and the formation of an inhibitory dopamine/Fe+3 complex, these findings suggest that a third mechanism exists by which dopamine (or DOPA) can inhibit Tyrosine Hydroxylase, adding further complexity to the regulation of catecholamine biosynthesis.
