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


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 


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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

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