IDENTIFICATION OF THE 4-AMINO ANALOG 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 trihydroxy-propyl s ide chains to recombinant rat neuronal nitric oxide (NO) synthase (EC 1.14.13.39) was determined by competition with 6R-[3'-H-3]-5,6,7,8-tet rahydro-L-erythro-biopterin (6R-[3'-H-3]H(4)biopterin). Although all b ut one ofthe derivatives exhibited only poor affinities (K-i 50 mu M), the 4-amino analogue of 6R-H(4)biopterin was a potent antagonist of 6 R-H(4)biopterin binding (K-i 13.2 nM). The 4-amino analogue of 6R-H(4) biopterin inhibited NO synthase stimulation by the natural cofactor BR -H(4)biopterin with an IC50 of 1 mu M without affecting the basal acti vity observed in the absence of added 6R-H(4)biopterin. Because the 4- amino analogue of 6R-H(4)biopterin also inhibited dihydropteridine red uctase (EC 1.6.99.7; IC50 20 mu M), our results support the hypothesis that redox cycling of H(4)biopterin might be required for the NO synt hase reaction.