Journal of Molecular Neuroscience

, Volume 4, Issue 2, pp 125–139

Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains

  • Paula Ribeiro
  • Yuehua Wang
  • Bruce A. Citron
  • Seymour Kaufman
Article

Abstract

The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains. A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified fromE. coli. The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity. On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme. These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455. Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparentKm for tyrosine and phenylalanine substrates, and a substantial increase in theKi for dopamine inhibition. The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition. The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor. These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.

Index Entries

Tyrosine hydroxylase mutagenesis dopamine regulation 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abate C. and Joh T. H. (1991) 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.,2, 203–215.PubMedGoogle Scholar
  2. Abate C., Smith J. A., and Joh T. H. (1988) Characterization of the catalytic domain of bovine adrenal tyrosine hydroxylase.Biochem. Biophys. Res. Comm. 151, 1446–1453.PubMedCrossRefGoogle Scholar
  3. Andersson K. K., Cox D. D., Que J. L., Flatmark T. and Haavik J. (1988) Resonance Raman studies on the blue-green-colored bovine adrenal tyrosine 3-monooxygenase (tyrosine hydroxylase).J. Biol. Chem. 263, 18,621–18,626.Google Scholar
  4. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72, 248–254.PubMedCrossRefGoogle Scholar
  5. Campbell D. G., Hardie D. G., and Vulliet P. R. (1986) Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase.J. Biol. Chem. 261, 10,489–10,492.Google Scholar
  6. Daubner S. C., Lauriano C., Haycock J. W., and Fitzpatrick P. F. (1992) Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase.J. Biol. Chem. 267, 12,639–12,646.Google Scholar
  7. Fitzpatrick P. F., Chlumsky L. J., Daubner S. C., and O'Malley K. L. (1990) Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.J. Biol. Chem. 265, 2042–2047.PubMedGoogle Scholar
  8. Frankel S., Sohn R., and Leinwand L. (1991) The use of sarkosyl in generating soluble protein after bacterial expression.Proc. Natl. Acad. Sci. USA,88, 1192–1196.PubMedCrossRefGoogle Scholar
  9. Grima B., Lamouroux A., Blanot F., Biguet N. F., and Mallet J. (1985) Complete coding sequence of rat tyrosine hydroxylase mRNA.Proc. Natl. Acad. Sci. USA.82, 617–621.PubMedCrossRefGoogle Scholar
  10. Haavik J., Andersson K. K., Petersson L., and Flatmark T. (1988) Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: large-scale purification and physicochemical properties.Biochim. Biophys. Acta 953, 142–156.PubMedGoogle Scholar
  11. Haavik J., Martinez A., and Flatmark T. (1990) pH-dependent release of catecholamines from tyrosine hydroxylase and the effect of phosphorylation of Ser-40.FEBS Lett. 262, 363–365.PubMedCrossRefGoogle Scholar
  12. Haycock J. W. and Haycock D. A. (1991) Tyrosine hydroxylase in rat brain dopaminergic nerve terminals.J. Biol. Chem. 266, 5650–5657.PubMedGoogle Scholar
  13. Hoeldtke R. and Kaufman S. (1977) Bovine adrenal tyrosine hydroxylase: purification and properties.J. Biol. Chem. 252, 3160–3169.PubMedGoogle Scholar
  14. Iwaki M., Phillips R. S., and Kaufman S. (1986) Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase.J. Biol. Chem. 261, 2051–2056.PubMedGoogle Scholar
  15. Joh T. H., Park D. H., and Reis D. J. (1978) Direct phosphorylation of brain tyrosine hydroxylase by cAMP-dependent protein kinases: A mechanism of enzyme activation.Proc. Natl. Acad. Sci. USA 75, 4744–4748.PubMedCrossRefGoogle Scholar
  16. Katz I., Lloyd T., and Kaufman S. (1976) Studies on phenylalanine and tyrosine hydroxylation by rat brain tyrosine hydroxylase.Biochem. Biophys. Res. Comm. 445, 567–578.Google Scholar
  17. Kaufman S. (1974) Properties of the pterin-dependent aromatic amino acid hydroxylases, inAromatic Amino Acids in the Brain. Ciba Foundation Symposium. Elsevier Excerpta Medica, North-Holland, Amsterdam, pp. 85–108.Google Scholar
  18. Kaufman S. (1985) Regulatory properties of phenylalanine, tyrosine and tryptophan hydroxylases.Biochem. Soc. Trans.,13, 433–436.PubMedGoogle Scholar
  19. Kaufman S. (1987) Aromatic amino acid hydroxylases, inThe Enzymes. Academic, Orlando, FL, pp. 217–282.Google Scholar
  20. Kaufman S. and Fisher D. B. (1974) Pterin-requiring aromatic amino acid hydroxylases, inMolecular Mechanisms of Oxygen Activation. Academic, New York, pp. 285–369.Google Scholar
  21. Kaufman S. and Kaufman E. E. (1985) Tyrosine hydroxylase, inFolates and Pterins, Chemistry and Biochemistry of Pterins. John Wiley, New York, pp. 251–352.Google Scholar
  22. Kuczenski R. (1973) Tyrosine hydroxylase: Activation by limited tryptic proteolysis.J. Biol. Chem. 248, 2261–2265.PubMedGoogle Scholar
  23. Lin K.-h. and Cheng S.-y. (1991) An efficient method to purify active eukaryotic proteins from the inclusion bodies inEscherichia coli.Biotechnology 11, 750.Google Scholar
  24. Liu X. and Vrana K. E. (1991) Leucine zippers and coiled-coils in the aromatic amino acid hydroxylases.Neurochem. Int. 1, 27–31.CrossRefGoogle Scholar
  25. Munson P. J. and Rodbard D. (1980) LIGAND: A versatile computerized approach for characterization of ligand-binding systems.Anal. Biochem. 107, 220–239.PubMedCrossRefGoogle Scholar
  26. Musacchio J. M., Wurtzburger R. J., and D'Angelo G. L. (1971) Different molecular forms of bovine adrenal tyrosine hydroxylase.Mol. Pharmacol. 7, 136–146.PubMedGoogle Scholar
  27. Nagatsu T., Levitt M., and Udenfriend S. (1964a) Tyrosine hydroxylase: the initial step in norepinephrine biosynthesis.J. Biol. Chem. 239, 2910–2917.PubMedGoogle Scholar
  28. Nagatsu T., Levitt M., and Udenfriend S. (1964b) A rapid and simple radioassay for tyrosine hydroxylase activity.Anal. Biochem. 9, 122–126.PubMedCrossRefGoogle Scholar
  29. Okuno S. and Fujisawa H. (1985) A new mechanism for regulation of tyrosine 3-monooxygenase by end product and cyclic AMP-dependent protein kinase.J. Biol. Chem. 260, 2633–2635.PubMedGoogle Scholar
  30. O'Neil R. R., Mitchell L. G., Merril C. R., and Rasband W. S. (1989) Use of image analysis to quantitate changes in form of mitochondrial DNA after X-irradiation.Appl. Theor. Electrophor. 1, 163–167.Google Scholar
  31. Petrack B., Sheppy F., and Fetzer V. (1968) Studies on tyrosine hydroxylase from bovine adrenal medulla.J. Biol. Chem. 243, 743–748.PubMedGoogle Scholar
  32. Ribeiro P., Pigeon D., and Kaufman S. (1991) The hydroxylation of phenylalanine and tyrosine by tyrosine hydroxylase from cultured pheochromocytoma cells.J. Biol. Chem. 266, 16,207–16,211.Google Scholar
  33. Ribeiro P., Wang Y., Citron B., and Kaufman S. (1992) The regulation of recombinant rat tyrosine hydroxylase by dopamine.Proc. Natl. Acad. Sci. USA 89, 9593–9597.PubMedCrossRefGoogle Scholar
  34. Sambrook J., Fritsch E. F., and Maniatis T. (1989) inMolecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp. 1.82–1.84.Google Scholar
  35. Sanger F., Nicklen S., and Coulson A. R. (1977) DNA sequencing with chain-terminating inhibitors.Proc. Natl. Acad. Sci. USA 74, 5463–5467.PubMedCrossRefGoogle Scholar
  36. Shiman R., Akino M., and Kaufman S. (1971) Solubilization and partial purification of tyrosine hydroxylase from bovine adrenal medulla.J. Biol. Chem. 246, 1330–1340.PubMedGoogle Scholar
  37. Studier F. W., Rosenberg A. H., Dunn J. J., and Dubendorff J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes.Meth. Enzymol. 185, 60–89.PubMedCrossRefGoogle Scholar
  38. Towbin H., Staehelin T., and Gordon J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. USA 76, 4350–4354.PubMedCrossRefGoogle Scholar
  39. Vigny A. and Henry J. P. (1981) Bovine adrenal tyrosine hydroxylase: comparative study of native and proteolyzed enzyme, and their interaction with anions.J. Neurochem. 36, 483–489.PubMedCrossRefGoogle Scholar
  40. Vulliet P. R., Langan T. A., and Weiner N. (1980) Tyrosine hydroxylase: A substrate for cyclic AMP-dependent protein kinase.Proc. Natl. Acad. Sci. USA 7, 92–96.CrossRefGoogle Scholar
  41. Wang Y.-H., Citron B., Ribeiro P., and Kaufman S. (1991) High-level expression of rat PC12 tyrosine hydroxylase cDNA inE. coli: Purification and characterization of the cloned enzyme.Proc. Natl. Acad. Sci. USA 88, 8779–8783.PubMedCrossRefGoogle Scholar
  42. Weiner N. Lee F. L., Dreyer E., and Barnes E. (1978) The activation of tyrosine hydroxylase in noradrnnergic neurons during acute nerve stimulation.Life Sci. 22, 1197–1216.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1993

Authors and Affiliations

  • Paula Ribeiro
    • 1
  • Yuehua Wang
    • 1
  • Bruce A. Citron
    • 1
  • Seymour Kaufman
    • 1
  1. 1.Laboratory of NeurochemistryNational Institute of Mental HealthBethesda

Personalised recommendations