A Flexible Loop in Tyrosine Hydroxylase Controls Coupling of Amino Acid Hydroxylation to Tetrahydropterin Oxidation

S. Colette Daubner, James Thomas McGinnis, Meredith Gardner, Stacie L. Kroboth, Adam R. Morris, Paul F Fitzpatrick

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected Ktyr values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/Ktyr to V/Kphe ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.

Original languageEnglish (US)
Pages (from-to)299-307
Number of pages9
JournalJournal of Molecular Biology
Volume359
Issue number2
DOIs
StatePublished - Jun 2 2006
Externally publishedYes

Fingerprint

Tyrosine 3-Monooxygenase
Hydroxylation
Phenylalanine Hydroxylase
Amino Acids
Alanine
Dihydroxyphenylalanine
Tyrosine
Substrate Specificity
Mixed Function Oxygenases
Site-Directed Mutagenesis
Mutagenesis
tetrahydropterin
Peptides

Keywords

  • alanine scanning
  • conformational change
  • iron-oxygen chemistry
  • substrate specificity
  • tyrosine hydroxylase

ASJC Scopus subject areas

  • Virology

Cite this

A Flexible Loop in Tyrosine Hydroxylase Controls Coupling of Amino Acid Hydroxylation to Tetrahydropterin Oxidation. / Colette Daubner, S.; McGinnis, James Thomas; Gardner, Meredith; Kroboth, Stacie L.; Morris, Adam R.; Fitzpatrick, Paul F.

In: Journal of Molecular Biology, Vol. 359, No. 2, 02.06.2006, p. 299-307.

Research output: Contribution to journalArticle

Colette Daubner, S. ; McGinnis, James Thomas ; Gardner, Meredith ; Kroboth, Stacie L. ; Morris, Adam R. ; Fitzpatrick, Paul F. / A Flexible Loop in Tyrosine Hydroxylase Controls Coupling of Amino Acid Hydroxylation to Tetrahydropterin Oxidation. In: Journal of Molecular Biology. 2006 ; Vol. 359, No. 2. pp. 299-307.
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