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

摘要

pTetrahydrobiopterin [(6iR/i)-5,6,7,8-tetrahydro-L-biopterin, Hsub4/subbiopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of iN/isup5/sup-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 Hsub4/subbiopterin-dependent reactions. Hsub4/subbiopterin substituted at iN/isup5/sup with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at iN/isup5/sup occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included iN/isup2/sup?-methyl Hsub4/subbiopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with iN/isup5/sup-methyl Hsub4/subbiopterin, iN/isup5/sup-formyl Hsub4/subbiopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, iN/isup5/sup-substituted derivatives were redox-active: iN/isup5/sup-methyl- and iN/isup5/sup-hydroxylmethyl Hsub4/subbiopterin, but not iN/isup5/sup-formyl- and iN/isup5/sup-acetyl Hsub4/subbiopterin, reduced 2,6-dichlorophenol indophenol. iN/isup5/sup-Substituted Hsub4/subbiopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and, depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: iN/isup5/sup-methyl- and iN/isup5/sup-hydroxymethyl Hsub4/subbiopterin inhibited phenylalanine hydroxylase, whereas iN/isup5/sup-formyl- and iN/isup5/sup-acetyl Hsub4/subbiopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by Hsub4/subbiopterin. They are compatible with a novel, non-classical, redox-active contribution of Hsub4/subbiopterin to the catalysis of the nitric oxide synthase reaction./p