Biochemical and Biophysical Research Communications
Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the l-tyrosine supply for melanogenesis in melanocytes
Section snippets
Materials and methods
6(R)-l-Erythro-5,6,7,8-tetrahydrobiopterin (6BH4) was obtained from Schircks Laboratories (Jona, Switzerland). All other reagents and chemicals were from Sigma (Poole/Dorset, UK). Recombinant human PAH was produced in the Department of Biochemistry and Molecular Biology, University of Bergen, and was a generous gift from Professor Aurora Martinez.
PAH enzyme assays. PAH activities were followed by measuring the formation of l-tyrosine from l-phenylalanine at 278 nm in Hepes buffer 0.02 M with 0.2 M
Recombinant PAH is activated by H2O2
Rates for PAH activity were determined in the presence of 0−5 × 10−3 M H2O2. Optimal activation occurred at concentrations >2.0 × 10−3 M (Fig. 1). First a kinetic analysis of the activation of PAH by 2.0 × 10−3 M H2O2 was performed in the presence of different concentrations of the substrate/activator l-phenylalanine. V vs S and Lineweaver–Burk analysis of these results showed a 4-fold increase in Vmax and a decrease in Km from 40 to 28 × 10−6 M with 2.0 × 10−3 M H2O2. The Lineweaver–Burk plot indicated mixed
Discussion
Over the past it was shown that H2O2 has a dual role in the control of pigmentation in the human epidermis [8], [13], [14], [21]. At low concentrations it activates several important enzymes that control melanogenesis, but at high concentrations it is a powerful inhibitor. H2O2 activates tyrosinase (EC 1.14.18.1) significantly at concentrations of 3 × 10−4 M, whereas the enzyme is deactivated in the presence of 10−3 M H2O2. The same ROS increases the transcription of GTP-cyclohydrolase I (GTP-CH-1,
Acknowledgments
This research was kindly supported by the University of Bradford, UK, Stiefel International, and German Deutsche Vitiligoverein, Hamburg.
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Present address: Department of Biomolecular Sciences, UMIST, P.O. Box 88, Manchester M90 1QD, UK.