Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine

S. Colette Daubner, Paul F. Fitzpatrick

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 Å, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the K(M) value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.

Original languageEnglish (US)
Pages (from-to)16440-16444
Number of pages5
JournalBiochemistry
Volume37
Issue number46
DOIs
StatePublished - Nov 17 1998

ASJC Scopus subject areas

  • Biochemistry

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