Regulatory Properties of the Tetrahydropterin Cofactor in the Reaction Catalysed by Human Tyrosine Hydroxylase Isoforms 1–4

  • B. Almås
  • D. Clement
  • T. Flatmark


The interaction of tyrosine hydroxylase (TH, EC, the rate-limiting enzyme of the catecholamine (CA) biosynthetic pathway, with its cofactor 6-(R)-5,6,7,8-tetrahydrobiopterin (BH4) seems to be highly specific. However, the dihydroxypropyl group in the Co-position of BH4 can be substituted with only moderate changes in kinetic parameters (1). TH is highly regulated, and many of the regulatory mechanisms (phosphorylation, feed-back inhibition) involve a change in the affinity for the cofactor. The four isoforms of human TH (hTH1–4) differ only in the length of their N-terminal regulatory domain (2). They all exist as homotetramers, containing a tetramerization domain in the C-terminal end. All isoforms have been expressed in E. coli and are isolated as nonphosphorylated apoenzymes with no CAs bound. The human recombinant enzyme thus represents a well defined system for structural and functional studies of the binding of BH4 to TH. We have previously reported a negative cooperativity in the binding of BH4 to hTH1 (Hill coefficient 0.39 < h < 0.58). This was modulated by phosphorylation by protein kinase A (h = 0.27 ± 0.03) (3). Time-course studies of the enzymatic reaction also demonstrated a time dependent change (burst) in activity, with a half-life of about 20 s for the kinetic transient (3). In this study we have further investigated the regulatory properties of BH4 with all human TH isoforms. Using synthetic cofactors as well as limited proteolysis, the relationship between the negative cooperativity and the kinetic transient in hTH1 was further characterised.


Tyrosine Hydroxylase Regulatory Property Hill Coefficient Limited Proteolysis Negative Cooperativity 
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  1. 1.
    Almås, B., Toska, K., Teigen, K., Groehn, V., Pfleiderer, W., Martinez, A., Flatmark, T., Haavik, J. A kinetic and conformational study on the interaction of tetrahydropteridines with tyrosine hydroxylase. Biochemistry 39: 13676–13686, 2000.PubMedCrossRefGoogle Scholar
  2. 2.
    Nagatsu, T., Ichinose, H. Comparative studies on the structure of tyrosine hydroxylase with those of the enzyme of various mammals. Comp. Biochem. Physiol. [C] 98: 203–210, 1991.CrossRefGoogle Scholar
  3. 3.
    Flatmark, T., Almas, B., Knappskog, P.M., Berge, S.V., Svebak, R.M., Chehin, R., Muga, A., Martínez, A. Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection. Eur. J. Biochem. 262: 1–11, 1999.CrossRefGoogle Scholar
  4. 4.
    Haavik, J., Le Bourdellés, B., Martínez, A., Flatmark, T., Mallet, J. Recombinant tyrosine hydroxylase isozymes: Reconstitution with iron and inhibitory effect of other metal ions. Eur. J. Biochem. 199: 371–378, 1991.PubMedCrossRefGoogle Scholar
  5. 5.
    McCulloch R.I., Fitzpatrick P.F. Limited proteolysis of tyrosine hydroxylase identifies residues 33–50 as conformationally sensitive to phosphorylation state and dopamine binding. Arch. Biochem. Biophys. 367: 143–145, 1999.PubMedCrossRefGoogle Scholar
  6. 6.
    Haycock, J.W. Four forms of tyrosine hydroxylase are present in human adrenal medulla. J. Neurochem. 56: 2139–2142, 1991.PubMedCrossRefGoogle Scholar
  7. 7.
    Alterio, J., Ravassard, P., Haavik, J., Le Caer, J-P., Biguet, N.F., Waksman, G., Mallet, J. Human tyrosine hydroxylase isoforms. Inhibition by excess tetrahydropterin and unusual behaviour of isoform 3 after cAMP-dependent protein kinase phosphorylation. J. Biol. Chem. 273: 10196–10201, 1998.PubMedCrossRefGoogle Scholar
  8. 8.
    Sutherland, C., Alterio, J., Campbell, D.G., Le Bourdellés, B., Mallet, J., Haavik, J., Cohen, P. Phosphorylation and activation of human tyrosine hydroxylase in vitro by mitogen-activated protein (MAP) kinase and MAP-kinase activated kinases 1 and 2. Eur. J. Biochem. 217: 715–722, 1993.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media New York 2002

Authors and Affiliations

  • B. Almås
    • 1
  • D. Clement
    • 1
  • T. Flatmark
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of BergenBergenNorway

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