Human phenylalanine hydroxylase is activated by H 2O 2: a novel mechanism for increasing the l-tyrosine supply for melanogenesis in melanocytes

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 H 2O 2, 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 V max for enzyme activity increased 4-fold by H 2O 2 (>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 Trp 120 residue to 5-OH-Trp 120 by H 2O 2 causing a conformational change in the regulatory domain. PAH was still activated by H 2O 2 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 H 2O 2 by UVB can activate epidermal PAH leading to an increased l-tyrosine pool for melanogenesis.