Human phenylalanine hydroxylase is activated by H2O2: a novel mechanism for increasing the l-tyrosine supply for melanogenesis in melanocytes

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Abstract

Epidermal phenylalanine hydroxylase (PAH) produces l-tyrosine from the essential amino acid l-phenylalanine supporting melanogenesis in human melanocytes. Those PAH activities increase linearly in the different skin phototypes I–VI (Fitzpatrick classification) and also increase up to 24 h after UVB light with only one minimal erythemal dose. Since UVB generates also H2O2, we here asked the question whether this reactive oxygen species could influence the activity of pure recombinant human PAH. Under saturating conditions with the substrate l-phenylalanine (1 × 10−3 M), the Vmax for enzyme activity increased 4-fold by H2O2 (>2.0 × 10−3 M). Lineweaver–Burk analysis identified a mixed activation mechanism involving both the regulatory and catalytic domains of PAH. Hyperchem molecular modelling and Deep View analysis support oxidation of the single Trp120 residue to 5-OH-Trp120 by H2O2 causing a conformational change in the regulatory domain. PAH was still activated by H2O2 in the presence of the electron donor/cofactor 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin despite slow oxidation of this cofactor. In vivo FT-Raman spectroscopy confirmed decreased epidermal phenylalanine in association with increased tyrosine after UVB exposure. Hence, generation of H2O2 by UVB can activate epidermal PAH leading to an increased l-tyrosine pool for melanogenesis.

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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.

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