title: Contrasting effects of N5-substituted tetrahydrobiopterin derivatives on phenylalanine hydroxylase, dihydropteridine reductase and nitric oxide synthase.
abstract: 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.
authors.affiliation: Institute for Medical Chemistry and Biochemistry of the University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria. ernst.r.werner@uibk.ac.at
authors.forename: E R
authors.initials: ER
authors.lastname: Werner
authors.forename: H J
authors.initials: HJ
authors.lastname: Habisch
authors.forename: A C
authors.initials: AC
authors.lastname: Gorren
authors.forename: K
authors.initials: K
authors.lastname: Schmidt
authors.forename: L
authors.initials: L
authors.lastname: Canevari
authors.forename: G
authors.initials: G
authors.lastname: Werner-Felmayer
authors.forename: B
authors.initials: B
authors.lastname: Mayer
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D004093:Dihydropteridine Reductase / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation; D019001:Nitric Oxide Synthase / Q000187:drug effects* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D010651:Phenylalanine Hydroxylase / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001708:Biopterins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D010651:Phenylalanine Hydroxylase; D004093:Dihydropteridine Reductase; C003402:sapropterin