Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.
Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-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 H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.
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Tetrahydrobiopterin nitric oxide N(5)-methyl N(5)-hydroxyl methyl H(4)biopterin N(5)-formyl N(5)-acetyl H(4)biopterin 2,6-dichlorophenol indophenol N(5)-Substituted H(4)biopterin dihydropteridine phenylalanine N(5)-methyl N(5)-hydroxymethyl H(4)biopterin phenylalanine N(5)-formyl N(5)-acetyl H(4)biopterin phenylalanine nitric oxide H(4)biopterin N(5) H(4)biopterin nitric oxide (6R)-5,6,7,8-tetrahydro-L-biopterin nitric oxide dihydropteridine phenylalanine H(4)biopterin H(4)biopterin N(5) methyl hydroxymethyl formyl acetyl N(5) H(4)biopterin phenylalanine dihydropteridine N(2)'-methyl H(4)biopterin N(5)-methyl H(4)biopterin N(5)-formyl H(4)biopterin nitric oxide N(5) dihydropteridine nitric oxide N5-substituted tetrahydrobiopterin phenylalanine nitric oxide synthases EC 1.14.13.39 dihydropteridine reductase phenylalanine hydroxylase phenylalanine hydroxylase phenylalanine hydroxylase nitric oxide synthase nitric oxide synthase rat neuronal nitric oxide synthase dihydropteridine reductase EC 1.6.99.7 phenylalanine hydroxylase EC 1.14.16.1 phenylalanine hydroxylase dihydropteridine reductase nitric oxide synthase dihydropteridine reductase nitric oxide synthase phenylalanine hydroxylase