Identification of the 4-amino analogue of tetrahydrobiopterin as a dihydropteridine reductase inhibitor and a potent pteridine antagonist of rat neuronal nitric oxide synthase

The binding of tetrahydropteridines with 6-di- and trihydroxypropyl side chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3´-3H]-5,6,7,8-tetrahydro-L-erythro-biopterin (6R-[3´-3H]H4biopterin). Although all but one of the derivatives exhibited only poor affinities (Ki 50 µM), the 4-amino analogue of 6R-H4biopterin was a potent antagonist of 6R-H4biopterin binding (Ki 13.2 nM). The 4-amino analogue of 6R-H4 biopterin inhibited NO synthase stimulation by the natural cofactor 6R-H4biopterin with an IC50 of 1 µM without affecting the basal activity observed in the absence of added 6R-H4biopterin. Because the 4-amino analogue of 6R-H4biopterin also inhibited dihydropteridine reductase (EC 1.6.99.7; IC50 20 µM), our results support the hypothesis that redox cycling of H4biopterin might be required for the NO synthase reaction.

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Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase

Biochemical Journal ◽

10.1042/bj3480579 ◽

2000 ◽

Vol 348 (3) ◽

pp. 579-583 ◽

Cited By ~ 5

Author(s):

Ernst R. WERNER ◽

Hans-Jörg HABISCH ◽

Antonius C. F. GORREN ◽

Kurt SCHMIDT ◽

Laura CANEVARI ◽

...

Keyword(s):

Nitric Oxide ◽

Nitric Oxide Synthase ◽

Phenylalanine Hydroxylase ◽

Neuronal Nitric Oxide Synthase ◽

Dihydropteridine Reductase ◽

Redox Active ◽

Competitive Inhibitors ◽

Oxide Synthase ◽

Active Contribution ◽

Stimulation Of

Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H4biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N5-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1), the well-studied classical H4biopterin-dependent reactions. H4biopterin substituted at N5 with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N5 occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N2ʹ-methyl H4biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N5-methyl H4biopterin, N5-formyl H4biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N5-substituted derivatives were redox-active: N5-methyl- and N5-hydroxylmethyl H4biopterin, but not N5-formyl- and N5-acetyl H4biopterin, reduced 2,6-dichlorophenol indophenol. N5-Substituted H4biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and, depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N5-methyl- and N5-hydroxymethyl H4biopterin inhibited phenylalanine hydroxylase, whereas N5-formyl- and N5-acetyl H4biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H4biopterin. They are compatible with a novel, non-classical, redox-active contribution of H4biopterin to the catalysis of the nitric oxide synthase reaction.

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