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

Abstract

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][716][717][718][719][720][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 identifiled 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.

Tetrahydro‐6‐biopterin is associated with tetrahydro‐7‐biopterin in primary murine mast cells

“…2). Ptefin 7-carboxylic acid is characterized by a marked reduction of its relative fluorescence intensity at 450 nm as compared to the fisomer [8]. In our experiments, it displayed only 9% of that produced by pterin.6.carboxylic acid.…”

“…Table I shows that H.,-7-biopterin averages 31,9 (+ 7,7)% of the total H~biopterin in the mast cells. This demonstrates that substantial amounts of this isomer do originate not only under in vitro phenylalanine hydroxylase assay conditions by omission of 4a-dehydratase [8] or in patients suffering from hyperphenylalaninemia who are supposed to be defective in this enzyme [6,7]. The data suggests that it rather originates also under in vivo conditions in primary mast cells during the course of the tryptophan 5-mono-oxygenase (EC 1.14.16.4) reaction which shares the same mechanism with the other aromatic amino acid hydroxylases.…”

“…The following pterins were used for reference: 6.biopterin, 7-biopterin; 6.neopterin, 7-ncopterln, monaptcrin, o-thrco.biopterin, 6-hydroxymethylpterin, 2-amino.4-oxopteridine, pterin.6.carboxaldehyde, pterin-6-carboxylic acid, and pterin-7.carboxylic acid, The following solvents were used: (I) 3% methanol, 1% aeetonitrile, pH 2,5 was adjusted with H.~PO~; (ll) 0.5% isopropanol, 0,5% methanol, pH 5.2 was adjusted with acetic acid; (lit) sodium acetate 50 mM, citric acid 5 raM, pH 5,2 was adjusted with NaOH; (IV) 5% methanol, sodium phosphate 100 raM° pH 3 was adjusted by H.~PO~, The flow rate was 1 ml.min -t at room temperature (20°C), For oxidation of pterins to thecorresponding carboxyLic acids (el. [18]) fractions 'bl' and "b2' were purified by HPLC in solvent I, concentrated by lyophilization and subjected to the alkaline permanganate method as described in [8],…”

“…In patients with a new variant of H4-6-biopterin deficiency and evident hyperphenylalaninemia the urinary excretion of 7-biopterin was increased and in some cases approached a 1:1 ratio as compared with 6-biopterin [6]. It had been strongly suggested [7] and then finally demonstrated [8] that the in vitro formation of 7-biopterin occurs via a non-enzymatic re-arrangement of 4a-hydroxy H~biop-terin which results from an opening of the pyrazine ring and its recyclization. A genetic defect is assumed to cause increased 7-biopterin levels in patients with hyperphenylalaninemia [7].…”

Tetrahydrobiopterin Deficiency: From Phenotype to Genotype

“…From in vitro experiments it is known that plimapterin can be formed during the PAH reaction in a phenylalanine-4-hydroxylase-stimulating protein (PHS)/pterin-4a-carbinolamine dehydratase (PCD) free system ( Fig. 2) (19,20). So far, all known enzymes involved in the biosynthesis or regeneration of BH4 in .…”