Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Oct 15;89(20):9593–9597. doi: 10.1073/pnas.89.20.9593

Regulation of recombinant rat tyrosine hydroxylase by dopamine.

P Ribeiro 1, Y Wang 1, B A Citron 1, S Kaufman 1
PMCID: PMC50178  PMID: 1357665

Abstract

Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.

Full text

PDF
9593

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abate C., Joh T. H. Limited proteolysis of rat brain tyrosine hydroxylase defines an N-terminal region required for regulation of cofactor binding and directing substrate specificity. J Mol Neurosci. 1991;2(4):203–215. [PubMed] [Google Scholar]
  2. Abate C., Smith J. A., Joh T. H. Characterization of the catalytic domain of bovine adrenal tyrosine hydroxylase. Biochem Biophys Res Commun. 1988 Mar 30;151(3):1446–1453. doi: 10.1016/s0006-291x(88)80524-2. [DOI] [PubMed] [Google Scholar]
  3. Andersson K. K., Cox D. D., Que L., Jr, Flatmark T., Haavik J. Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase). Evidence that the feedback inhibitors adrenaline and noradrenaline are coordinated to iron. J Biol Chem. 1988 Dec 15;263(35):18621–18626. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Davis M. D., Kaufman S., Milstien S. A modified ferrozine method for the measurement of enzyme-bound iron. J Biochem Biophys Methods. 1986 Aug;13(1):39–45. doi: 10.1016/0165-022x(86)90006-0. [DOI] [PubMed] [Google Scholar]
  6. Haavik J., Andersson K. K., Petersson L., Flatmark T. Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties. Biochim Biophys Acta. 1988 Mar 23;953(2):142–156. doi: 10.1016/0167-4838(88)90019-2. [DOI] [PubMed] [Google Scholar]
  7. Haavik J., Le Bourdelles B., Martinez A., Flatmark T., Mallet J. Recombinant human tyrosine hydroxylase isozymes. Reconstitution with iron and inhibitory effect of other metal ions. Eur J Biochem. 1991 Jul 15;199(2):371–378. doi: 10.1111/j.1432-1033.1991.tb16133.x. [DOI] [PubMed] [Google Scholar]
  8. Haavik J., Martínez A., Flatmark T. pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser-40. FEBS Lett. 1990 Mar 26;262(2):363–365. doi: 10.1016/0014-5793(90)80230-g. [DOI] [PubMed] [Google Scholar]
  9. Ichikawa S., Nasrin S., Nagatsu T. Expression of mouse tyrosine hydroxylase in Escherichia coli. Biochem Biophys Res Commun. 1991 Jul 31;178(2):664–671. doi: 10.1016/0006-291x(91)90159-5. [DOI] [PubMed] [Google Scholar]
  10. Ikeda M., Fahien L. A., Udenfriend S. A kinetic study of bovine adrenal tyrosine hydroxylase. J Biol Chem. 1966 Oct 10;241(19):4452–4456. [PubMed] [Google Scholar]
  11. Le Bourdellès B., Horellou P., Le Caer J. P., Denèfle P., Latta M., Haavik J., Guibert B., Mayaux J. F., Mallet J. Phosphorylation of human recombinant tyrosine hydroxylase isoforms 1 and 2: an additional phosphorylated residue in isoform 2, generated through alternative splicing. J Biol Chem. 1991 Sep 15;266(26):17124–17130. [PubMed] [Google Scholar]
  12. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  13. NAGATSU T., LEVITT M., UDENFRIEND S. A RAPID AND SIMPLE RADIOASSAY FOR TYROSINE HYDROXYLASE ACTIVITY. Anal Biochem. 1964 Sep;9:122–126. doi: 10.1016/0003-2697(64)90092-2. [DOI] [PubMed] [Google Scholar]
  14. NAGATSU T., LEVITT M., UDENFRIEND S. TYROSINE HYDROXYLASE. THE INITIAL STEP IN NOREPINEPHRINE BIOSYNTHESIS. J Biol Chem. 1964 Sep;239:2910–2917. [PubMed] [Google Scholar]
  15. Okuno S., Fujisawa H. A new mechanism for regulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase. J Biol Chem. 1985 Mar 10;260(5):2633–2635. [PubMed] [Google Scholar]
  16. Ribeiro P., Pigeon D., Kaufman S. The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells. J Biol Chem. 1991 Aug 25;266(24):16207–16211. [PubMed] [Google Scholar]
  17. Shiman R., Akino M., Kaufman S. Solubilization and partial purification of tyrosine hydroxylase from bovine adrenal medulla. J Biol Chem. 1971 Mar 10;246(5):1330–1340. [PubMed] [Google Scholar]
  18. Udenfriend S., Zaltzman-Nirenberg P., Nagatsu T. Inhibitors of purified beef adrenal tyrosine hydroxylase. Biochem Pharmacol. 1965 May;14(5):837–845. doi: 10.1016/0006-2952(65)90103-6. [DOI] [PubMed] [Google Scholar]
  19. Wang Y. H., Citron B. A., Ribeiro P., Kaufman S. High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8779–8783. doi: 10.1073/pnas.88.19.8779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Witt J. J., Roskoski R., Jr Rapid protein kinase assay using phosphocellulose-paper absorption. Anal Biochem. 1975 May 26;66(1):253–258. doi: 10.1016/0003-2697(75)90743-5. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES