Conversion of 6-substituted tetrahydropterins to 7-isomers via phenylalanine hydroxylase-generated intermediates

A new variant form of hyperphenylalaninemia has recently been discovered in which the patients characteristically excrete 7-biopterin in their urines in addition to the natural 6-biopterin (Curtius, H. Ch., Kuster, T., Matasovic, A., Blau, N. & Dhondt, J.-L. (1988) Biochem. Biophys. Res. Commun. 153, 715-721). This isomer had not been found previously in humans, and although its origin was not established, preliminary evidence suggested that it might be produced from 6-biopterin. We have now found that 7-biopterin can be formed in vitro from (6R)-tetrahydrobiopterin during the hydroxylation of phenylalanine catalyzed by phenylalanine hydroxylase [L-phenylalanine, tetrahydrobiopterin:oxygen oxidoreductase (4-hydroxylating), EC 1.14.16.1]. The resulting 7-biopterin was unequivocally identified by the following criteria: preparative isolation and conversion to 7-hydroxymethylpterin following periodate oxidation and borohydride reduction, quantitative conversion to pterin-7-carboxylic acid after oxidation with permanganate, and liquid chromatography/thermospray mass spectrometry. Addition of 4a-carbinolamine dehydratase, an enzyme involved in the regeneration of tetrahydrobiopterin from the pterin carbinolamine intermediate (also called 4a-hydroxytetrahydrobiopterin) formed in the phenylalanine hydroxylase reaction, greatly decreased the amount of the 7-biopterin formed. This result implies that the in vitro formation of 7-biopterin occurs via the nonenzymatic rearrangement of the unstable substrate of the dehydratase, 4a-hydroxytetrahydrobiopterin, and suggests that this new variant of hyperphenylalaninemia may be caused by a lack of 4a-carbinolamine dehydratase activity. A mechanism for the rearrangement is proposed that predicts that other 6-substituted tetrahydropterin substrates of the aromatic amino acid hydroxylases could also give rise to rearranged products from an opening of the pyrazine ring of the corresponding 4a-hydroxytetrahydropterin intermediate.