Oxygen-18 kinetic isotope effect studies of the tyrosine hydroxylase reaction: Evidence of rate limiting oxygen activation

Wilson A. Francisco, Gaochao Tian, Paul F. Fitzpatrick, Judith P. Klinman

Research output: Contribution to journalArticle

66 Scopus citations

Abstract

Tyrosine hydroxylase converts tyrosine to dihydroxyphenylalanine utilizing a tetrahydropterin cofactor and molecular oxygen. Previous deuterium isotope effect studies have raised the possibility that the activation of oxygen might be the rate-limiting step for this reaction. To test the validity of this proposal, we have measured the 18O kinetic isotope effects for the tyrosine hydroxylase reaction as a function of amino acid substrate, tetrahydropterin derivative, and pH. The measured 18O isotope effects are nearly constant in every condition tested with an average value of 1.0175 ± 0.0019. These results are consistent with a change in the bond order to oxygen in the rate determining step. Furthermore, the isotope effects measured with the coupled substrate 4-methoxyphenylalanine and the completely uncoupled substrate 4-aminophenylalanine are identical, implying the same rate determining step independent of whether oxygen activation is coupled to substrate hydroxylation. The results of these studies provide strong support for a rate limiting reductive activation of molecular oxygen, most likely via a one-electron transfer from the tetrahydropterin to form superoxide anion as the first reactive intermediate.

Original languageEnglish (US)
Pages (from-to)4057-4062
Number of pages6
JournalJournal of the American Chemical Society
Volume120
Issue number17
DOIs
StatePublished - May 6 1998

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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