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

Sura, Giri R.; Daubner, S. Colette; Fitzpatrick, Paul F.

doi:10.1111/j.1471-4159.2004.02742.x

Cited by 5 publications

(6 citation statements)

References 23 publications

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“…Tissue-specific alternative splicing of the H 4 -biopterin-requiring enzyme tyrosine hydroxylase is proposed to allow differential regulation through dopamine binding and cAMP-dependent phosphorylation in Drosophila [27], but human isoforms show no effect of phosphorylation on dopa binding [28]. N-terminal splice variants of GCH with distinct expression patterns have been characterized in Drosophila [29]; however, they cannot be compared with mammalian GCH, which lacks the N-terminal extension present in Drosophila isoforms.…”

Section: Discussionmentioning

confidence: 99%

Interaction of human GTP cyclohydrolase I with its splice variants

Pandya

Golderer

Werner

et al. 2006

Biochemical Journal

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Tetrahydrobiopterin is an essential cofactor for aromatic amino acid hydroxylases, ether lipid oxidase and nitric oxide synthases. Its biosynthesis in mammals is regulated by the activity of the homodecameric enzyme GCH (GTP cyclohydrolase I; EC 3.5.4.16). In previous work, catalytically inactive human GCH splice variants differing from the wild-type enzyme within the last 20 C-terminal amino acids were identified. In the present study, we searched for a possible role of these splice variants. Gel filtration profiles of purified recombinant proteins showed that variant GCHs form high-molecular-mass oligomers similar to the wild-type enzyme. Co-expression of splice variants together with wild-type GCH in mammalian cells revealed that GCH levels were reduced in the presence of splice variants. Commensurate with these findings, the GCH activity obtained for wild-type enzyme was reduced 2.5-fold through co-expression with GCH splice variants. Western blots of native gels suggest that splice variants form decamers despite C-terminal truncation. Therefore one possible explanation for the effect of GCH splice variants could be that inactive variants are incorporated into GCH heterodecamers, decreasing the enzyme stability and activity.

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Section: Discussionmentioning

confidence: 99%

Interaction of human GTP cyclohydrolase I with its splice variants

Pandya

Golderer

Werner

et al. 2006

Biochemical Journal

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“…Construction of human TyrH isoform 1 cDNA inserted into pET23d was as described in Sura et al16 Plasmids for expression of the mutant proteins were constructed using the Stratagene QuikChange site‐directed mutagenesis method with the following primers: T245P, 5′‐GGCCTCTACGCC CCG CACGCCTGCGG‐3′; T283M, 5′‐CCTGAAGGAGCGC ATG GGCTTCCAGCTGCG‐3′; R306H, 5′‐CCAGCCTGGCCTTC CAC GTGTTCCAGTGCACC‐3′; and T463M, 5′‐CGACCCGTAC ATG CTGGCCATCGACG‐3′. The mutated triplet is indicated in bold in each case.…”

Section: Methodsmentioning

confidence: 99%

Effects of mutations in tyrosine hydroxylase associated with progressive dystonia on the activity and stability of the protein

Royo¹,

Daubner²,

Fitzpatrick³

2004

Proteins

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Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine. Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31). All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M. To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified. Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme. The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50%. In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations. The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme. The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value. The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities.

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“…The ferrous enzyme is readily oxidized to the ferric form , , and catecholamines bind to the ferric enzyme with dissociation constants of ∼1 nM , . Phosphorylation of Ser40 activates TyrH by increasing the rate constant for dissociation of catecholamines ∼500-fold − . This allows tetrahydrobiopterin to reduce the iron, reactivating the enzyme .…”

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confidence: 99%

Identification by Hydrogen/Deuterium Exchange of Structural Changes in Tyrosine Hydroxylase Associated with Regulation

Wang¹,

Sura²,

Dangott

et al. 2009

Biochemistry

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The activity of tyrosine hydroxylase is regulated by reversible phosphorylation of serine residues in an N-terminal regulatory domain and catecholamine inhibition at the active site. Catecholamines such as dopamine bind very tightly to the resting enzyme; phosphorylation of Ser40 decreases the affinity for catecholamines by three orders of magnitude. The effects of dopamine binding and phosphorylation of Ser40 on the kinetics of deuterium incorporation into peptide bonds were examined by mass spectrometry. When dopamine is bound, three peptic peptides show significantly slower deuterium incorporation, 35-41 and 42-71 in the regulatory domain and 295-299 in the catalytic domain. In the phosphorylated enzyme, peptide 295-299 shows more rapid incorporation of deuterium, while 35-41 and 42-71 can not be detected. These results are consistent with tyrosine hydroxylase existing in two different conformations. In the closed conformation, the regulatory domain lies across the active site loop containing residues 295-298; this is stabilized when dopamine is bound in the active site. In the open conformation, the regulatory domain has moved out of the active site, allowing substrates access; this conformation is favored by phosphorylation of Ser40.Tyrosine hydroxylase (TyrH) catalyzes the first and rate-limiting step of catecholamine biosynthesis, the conversion of tyrosine into dihydroxyphenylalanine, utilizing a tetrahydropterin as the source of electrons. The enzyme belongs to the small family of aromatic amino acid hydroxylases, which also includes phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) (1). All three enzymes play critical physiological roles; PheH is responsible for catabolism of excess phenylalanine in the diet, while TrpH is the first and rate-limiting enzyme in serotonin biosynthesis. The mammalian forms of these enzymes are homotetramers (2-4) in which each monomer contains a regulatory domain of 100-150 amino acids at the N-terminus and a larger catalytic domain of around 350 amino acids at the Cterminus (5-9). The homologous (5) catalytic domains contain all of the residues required for catalysis and for substrate specificity (10), while the regulatory domains exhibit low levels of sequence identity (5,11). Structures have been determined for the catalytic domains of all three enzymes (12)(13)(14), but the only regulatory domain with an available structure is that of PheH *Address correspondence to: Paul F. Fitzpatrick Department of Biochemistry, MC 7760 University of Texas Health Science Center at San Antonio San Antonio, TX 78229-3900 fitzpatrick@biochem.uthscsa.edu ph: 210-567-8264; fax: 210-567-8778. 1 Abbreviations used: TyrH, rat tyrosine hydroxylase; PheH, phenylalanine hydroxylase; TrpH, tryptophan hydroxylase; PKA, protein kinase A. Supporting Information Available:The effects of dopamine and phosphorylation on the kinetics of deuterium incorporation into all of the peptic peptides for TyrH. This material is available free of charge via the Internet at http://pubs.ac...

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

Abstract: There was an error in Table 4 of the above article which was published in J. Neurochem. 90, pp 970-978. The values for K d were given in lM, however, this should have been nM. Furthermore, incorrect reference to footnotes were cited. The publishers apologise for any inconvenience caused.

Cited by 5 publications

References 23 publications

Interaction of human GTP cyclohydrolase I with its splice variants

Interaction of human GTP cyclohydrolase I with its splice variants

Effects of mutations in tyrosine hydroxylase associated with progressive dystonia on the activity and stability of the protein

Identification by Hydrogen/Deuterium Exchange of Structural Changes in Tyrosine Hydroxylase Associated with Regulation

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