Reductive Activation of Phenylalanine Hydroxylase and Its Effect on the Redox State of the Non-Heme Iron

D. E. Wallick, L. M. Bloom, S. J. Benkovic, Betty Jean Gaffney

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Abstract

Phenylalanine hydroxylase undergoes an obligatory prereduction step in order to become catalytically active as shown by stopped-flow kinetics and by measuring tyrosine formation at limiting 6-methyltetrahydropterin levels. This initial step requires oxygen and involves conversion of 6-methyltetrahydropterin directly to the quinonoid form with or without phenylalanine. The EPR spectrum of the resting enzyme (geff = 9.4-8.7, 4.3 and geff = 6.7, 5.4) is consistent with two species possessing distinctively different ligand environments for the non-heme, high-spin Fe3+. The intensity of the geff ⋍ 4.3 feature is inversely proportional to the specific activity of the enzyme, whereas the intensity of the geff ⋍ 6.7-S.4 region correlates with the activity of the enzyme. The latter features are lost upon addition of phenylalanine under anaerobic or aerobic conditions. In the presence of o-phenanthroline, the operation of the prereduction step results in nearly quantitative trapping of the iron in an Fe2+ redox state. Dithionite can substitute for 6-methyltetrahydropterin in an anaerobic prereduction step, generating a catalytically active phenylalanine hydroxylase containing Fe2+ that functions aerobically to produce tyrosine from added 6-methyltetrahydropterin in a 1/1 stoichiometry. Reductive titration of the hydroxylase by dithionite is consistent with the addition of one electron/subunit for coupled turnover. The implications of these findings for the mechanism of action of this enzyme are briefly discussed.

Original languageEnglish (US)
Pages (from-to)1295-1302
Number of pages8
JournalBiochemistry
Volume23
Issue number6
DOIs
StatePublished - Mar 1984

All Science Journal Classification (ASJC) codes

  • Biochemistry

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