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Journal of Molecular Neuroscience

, Volume 9, Issue 1, pp 35–48 | Cite as

A chimeric tyrosine/tryptophan hydroxylase

The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity
  • Susan M. Mockus
  • Sean C. Kumer
  • Kent E. Vrana
Original Articles

Abstract

The neurotransmitter biosynthetic enzymes, tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH) are each composed of an amino-terminal regulatory domain and a carboxylterminal catalytic domain. A chimeric hydroxylase was generated by coupling the regulatory domain of TH (TH-R) to the catalytic domain of TPH (TPH-C) and expressing the recombinant enzyme in bacteria. The chimeric junction was created at proline 165 in TH and proline 106 in TPH because this residue is within a conserved five amino-acid span (ValProTrpPhePro) that defines the beginning of the highly homologous catalytic domains of TH and TPH. Radioenzymatic activity assays demonstrated that the TH-R/TPH-C chimera hydroxylates tryptophan, but not tyrosine. Therefore, the regulatory domain does not confer substrate specificity. Although the TH-R/TPH-C enzyme did serve as a substrate for protein kinase (PKA), activation was not observed following phosphorylation. Phosphorylation studies in combination with kinetic data provided evidence that TH-R does not exert a dominant influence on TPH-C. Stability assays revealed that, whereas TH exhibited a t1/2 of 84 min at 37°C, TPH was much less stable (t 1/2=28.3 min). The stability profile of TH-R/TPH-C, however, was superimposable on that of TH. Removal of the regulatory domain (a deletion of 165 amino acids from the N-terminus) of TH rendered the catalytic domain highly unstable, as demonstrated by at 1/2 of 14 min. The authors conclude that the regulatory domain of TH functions as a stabilizer of enzyme activity. As a corollary, the well-characterized instability of TPH may be attributed to the inability of its regulatory domain to stabilize the catalytic domain.

Index Entries

Chimera phosphorylation tryptophan hydroxylase tyrosine hydroxylase bacterial expression 

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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. Commun. 151, 1446–1453.PubMedCrossRefGoogle Scholar
  3. Ausubel F. M., Brent R., Kingston R. E., Moore D., Seidman J. G., Smith J. A., and Struhl K. (1987)Current Protocols in Molecular Biology, Wiley, New York.Google Scholar
  4. Beevers S. J., Knowles R. G., and Pogson C. I. (1983) A sensitive radiometric assay for tryptophan hydroxylase applicable to crude extracts.J. Neurochem. 40, 894–897.PubMedCrossRefGoogle Scholar
  5. Bonnefoy E., Ferrara P., Rohrer H., Gros F., and Thibault J. (1988) Role of the N-terminus of rat pheochromocytoma tyrosine hydroxylase in the regulation of the enzyme's activity.Eur. J. Biochem. 174, 685–690.PubMedCrossRefGoogle Scholar
  6. Cash C. D. P., Vayer P., Mandel P., and Maitre M. (1985) Tryptophan 5-hydroxylase: rapid purification from whole brain and production of specific antiserum.Eur. J. Biochem. 149, 239–245.PubMedCrossRefGoogle Scholar
  7. Cotton R. G. H., McAdam W., Jennings I., and Morgan F. J. (1988) A monoclonal antibody to aromatic amino acid hydroxylases: identification of the epitope.Biochem. J. 255, 193–196.PubMedGoogle Scholar
  8. Daubner S. C., Lohse D. L., and Fitzpatrick P. F. (1993) Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.Protein Sci. 2, 1452–1460.PubMedGoogle Scholar
  9. Daubner S. C. and Fitzpatrick P. F. (1993) Lysine 241 of tyrosine hydroxylase is not required for binding of tetrahydrobiopterin substrate.Arch. Biochem. Biophys. 302, 455–460.PubMedCrossRefGoogle Scholar
  10. Daubner S. C. and Piper M. M. (1995) Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.Protein Sci. 4, 538–541.PubMedGoogle Scholar
  11. D'Sa C. M., Arthur R. E. Jr., and Kuhn D. M. (1996) Expression and deletion mutagenesis of tryptophan hydroxylase fusion proteins: delineation of the enzyme catalytic core.J. Neurochem. 67, 917–926.PubMedCrossRefGoogle Scholar
  12. Edelman A. M., Raese J. D., Lazar M. A., and Barchas J. D. (1981) Tyrosine hydroxylase: studies on the phosphorylation of a purified preparation of the brain enzyme by cyclic AMP-dependent protein kinase.J. Pharmacol. Exp. Ther. 216, 647–653.PubMedGoogle Scholar
  13. Ehret M., Cash C. D., Hamon M., and Maitre M. (1989) Partial demonstration of the phosphorylation of rat brain tryptophan hydroxylase by Ca2+/ calmodulin-dependent protein kinase.J. Neurochem. 52, 1886–1891.PubMedCrossRefGoogle Scholar
  14. Friedman P. A., Kappelman A. H., and Kaufman S. (1972) Partial purification and characterization of tryptophan hydroxylase from rabbit hindbrain.J. Biol. Chem. 247, 4165–4173.PubMedGoogle Scholar
  15. Fujisawa H. and Nakata H. (1987) Tryptophan 5-monooxygenase from mouse mastocytoma clone P815.Methods in Enzymol. 142, 93–96.CrossRefGoogle Scholar
  16. Furukawa Y., Ikuta N., Omata S., Yamauchi T., Isobe T., and Ichimura T. (1993) Demonstration of the phosphorylation-dependent interaction of tryptophan hydroxylase with the 14-3-3 protein.Biochem. Biophys. Res. Commun. 194, 144–149.PubMedCrossRefGoogle Scholar
  17. Grenett H. E., Ledley F. D., Reed L. L., and Woo S. L. C. (1987) Full-length cDNA for rabbit tryptophan hydroxylase: functional domains and evolution of aromatic amino acid hydroxylases.Proc. Natl. Acad. Sci. USA 84, 5530–5534.PubMedCrossRefGoogle Scholar
  18. 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
  19. Huang D. and Amero S. A. (1997) Measurement of antigen by enhanced chemiluminescent western blot.BioTechniques 22, 454–458.PubMedGoogle Scholar
  20. Hufton S. E., Jennings I. G., and Cotton R. G. H. (1995) Structure and function of the aromatic amino acids hydroxylases.J. Biochem. 311, 353–366.Google Scholar
  21. Jequier E., Robinson D. S., Lovenberg W., and Sjoerdsma A. (1969) Further studies on tryptophan hydroxylase in rat brainstem and beef pineal.Biochem. Pharmacol. 18, 1071–1081.PubMedCrossRefGoogle Scholar
  22. Johansen P. A., Jennings I. G., Cotton R. G. H., and Kuhn D. M. (1995) Tryptophan hydroxylase is phosphorylated by protein kinase A.J. Neurochem. 65, 882–888.PubMedCrossRefGoogle Scholar
  23. Johansen P. A., Jennings I. G., Cotton R. G. H., and Kuhn D. M. (1996) Phosphorylation and activation of tryptophan hydroxylase by exogenous protein kinase A.J. Neurochem. 66, 817–823.PubMedCrossRefGoogle Scholar
  24. Joh T. H., Park D. H., and Reis D. J. (1978) Direct phosphorylation of brain tyrosine hydroxylase by cyclic AMP-dependent protein kinase: mechanism of enzyme activation.Proc. Natl. Acad. Sci. USA 75, 4744–4746.PubMedCrossRefGoogle Scholar
  25. Joh T. H., Shikimi T., Pickel V. M., and Reis D. J. (1975) Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain.Proc. Natl. Acad. Sci. USA 72, 3575–3579.PubMedCrossRefGoogle Scholar
  26. Kuhn D. M., Ruskin B., and Lovenberg W. (1980) Tryptophan hydroxylase: role of oxygen, iron, sulfhydryl groups as determinants of stability and catalytic activity.J. Biol. Chem. 255, 4137–4143.PubMedGoogle Scholar
  27. Kuhn D. M., Arthur R. A. Jr., and States J. C. (1997) Phosphorylation and activation of brain tryptophan hydroxylase: identification of serine-58 as a substrate site for protein kinase A.J. Neurochem. 68, 2220–2223.PubMedCrossRefGoogle Scholar
  28. Kumer S. C., Mockus S. M., Rucker P. J., and Vrana K. E. (1997) Amino terminal deletion analysis of tryptophan hydroxylase: PKA phosphorylation occurs at serine-58.J. Neurochem., in press.Google Scholar
  29. Kumer S. C. and Vrana K. E. (1996) The intricate regulation of tyrosine hydroxylase activity and gene expression.J. Neurochem. 67, 443–462.PubMedCrossRefGoogle Scholar
  30. Laemmli U. K. (1970) Cleavage of structural protein during the assembly of the head of bacteriophage T4.Nature 227, 680–685.PubMedCrossRefGoogle Scholar
  31. Ledley F. D., DiLella A. G., Kwok S. C. M., and Woo S. L. C. (1985) Homology between phenylalanine and tyrosine hydroxylase reveals common structural and functional domains.Biochemistry 24, 3389–3394.PubMedCrossRefGoogle Scholar
  32. Levitt M., Spector S., Sjoerdsma A., and Udenfriend S. (1965) Elucidation of the rate-limiting step in norepinephrine biosynthesis of the perfused guineapig heart.J. Pharmacol. Ther. 148, 1–7.Google Scholar
  33. Liu X. and Vrana K. E. (1991) Leucine zippers and coiled-coils in the aromatic amino acid hydroxylases.Neurochem. Int. 18, 27–31.CrossRefPubMedGoogle Scholar
  34. Lohse D. L. and Fitzpatrick P. F. (1993) Identification of the intersubunit binding region in rat tyrosine hydroxylase.Biochem. Biophys. Res. Commun. 197, 1543–1548.PubMedCrossRefGoogle Scholar
  35. Makita Y., Okuno S., and Fujisawa H. (1990) Involvement of activator protein in the activation of tryptophan hydroxylase by cAMP-dependent protein kinase.FEBS Lett. 268, 185–188.PubMedCrossRefGoogle Scholar
  36. Mockus S. M., Kumer S. C., and Vrana K. E. (1997) Carboxyl terminal deletion analysis of tryptophan hydroxylase.Biochim. Biophys. Acta, in press.Google Scholar
  37. Nakata H. and Fujisawa H. (1982a) Purification and properties of tryptophan 5-monoxygenase from rat brainstem.Eur. J. Biochem. 122, 41–47PubMedCrossRefGoogle Scholar
  38. Nakata H. and Fujisawa H. (1982b) Tryptophan 5-monoxygenase from mouse mastocytoma.Eur. J. Biochem. 124, 595–601.PubMedCrossRefGoogle Scholar
  39. Nukiwa T., Tohyama C., Okita T., and Ichiyama A. (1982) Purification and some properties of bovine pineal tryptophan 5-monooxygenase.Biochem. Biophys. Res. Commun. 60, 1029–1035.CrossRefGoogle Scholar
  40. O'Neill 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.PubMedGoogle Scholar
  41. Ota A., Yoshida S., and Nagatsu T. (1995) Deletion mutagenesis of human tyrosine hydroxylase type 1 regulatory domain.Biochem. Biophys. Res. Commun. 213, 1099–1106.PubMedCrossRefGoogle Scholar
  42. Quinsey N. S., Lenaghan C. M., and Dickson P. W. (1996) Identification of Gln313 and Pro327 as residues critical for substrate inhibition in tyrosine hydroxylase.J. Neurochem. 66, 908–914.PubMedCrossRefGoogle Scholar
  43. Reinhard J., Smith G., and Nichol C. (1986) A rapid and sensitive assay for tyrosine-3-monooxygenase based upon the release of3H2O and adsorption of [3H] tyrosine by charcoal.Life Sci. 39, 2185–2189.PubMedCrossRefGoogle Scholar
  44. Ribeiro P., Wang Y., Citron B. A., and Kaufman S. (1993) Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains.J. Mol. Neurosci. 4, 125–139.PubMedCrossRefGoogle Scholar
  45. Sambrook J., Fritsch E. F., and Maniatis T. (1989)Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  46. Tipper P. T., Citron B. A., Ribeiro P., and Kaufman S. (1994) Cloning and expression of rabbit and human brain tryptphan hydroxylase cDNA inEscherichia coli.Arch. Biochem. Biophys.,315, 445–453.PubMedCrossRefGoogle Scholar
  47. Vitto A. and Mandell A. J. (1981) Stability properties of activated tryptophan hydroxylase from rat midbrain.J. Neurochem. 37, 601–607.PubMedCrossRefGoogle Scholar
  48. Vrana S. L., Dworkin S. I., and Vrana K. E. (1993) Radioenzymatic assay for tryptophan hydroxylase: [3H]H2O release assessed by charcoal adsorption.J. Neurosci. Meth.,48, 123–129.CrossRefGoogle Scholar
  49. Vrana K. E., Rucker P. J., and Kumer S. C. (1994a) Recombinant rabbit tryptophan hydroxylase is a substrate for cAMP-dependent protein kinase.Life Sci. 55, 1045–1052.PubMedCrossRefGoogle Scholar
  50. Vrana K. E., Walker S. J., Rucker P., and Liu X. (1994b) A carboxyl terminal leucine zipper is required for tyrosine hydroxylase tetramer formation.J. Neurochem. 63, 2014–2020.PubMedCrossRefGoogle Scholar
  51. Vulliet P. R., Langan T. A., and Weiner N. (1980) Tyrosine hydroxylase: a substrate of cyclic AMP-dependent protein kinase.Proc. Natl. Acad. Sci. USA 77, 92–96.PubMedCrossRefGoogle Scholar
  52. Walker S. J., Liu X., Roskoski R. Jr., and Vrana K. E. (1994) Catalytic core of rat tyrosine hydroxylase: terminal deletion analysis of a bacterially-expressed enzyme.Biochim. Biophys. Acta 1206, 113–119.PubMedGoogle Scholar
  53. 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
  54. Widmer F., Mutus, B., RamaMurthy J., Snieckus V. A., and Viswanatha, T. (1975) Partial purification of rabbit hind brain tryptophan hydroxylase by affinity chromatography.Life Sci. 17, 1297–1302.PubMedCrossRefGoogle Scholar
  55. Yamauchi T. and Fujisawa H. (1979) Regulation of bovine adrenal tyrosine-3-monooxygenase by phosphorylation-dephosphorylation reaction catalyzed by adenosine 3′,5′-monophosphate-dependent protein kinase and phosphoprotein phosphatase.J. Biol. Chem. 254, 6408–6413.PubMedGoogle Scholar
  56. Yang X. J. and Kaufman S. (1994) High-level expression and deletion mutagenesis of human tryptophan hydroxylase.Proc. Natl. Acad. Sci. USA 91, 6659–6663.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 1997

Authors and Affiliations

  • Susan M. Mockus
    • 1
  • Sean C. Kumer
    • 2
  • Kent E. Vrana
    • 2
  1. 1.Program in Neuroscience, Bowman Gray School of MedicineWake Forest UniversityWinston-Salem
  2. 2.Department of Physiology and Pharmacology, Bowman Gray School of MedicineWake Forest UniversityWinston-Salem

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