Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase

The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO syntha ses (NOS), For unknown reasons, all NOS co-purify with substoichiometric am ounts of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)Bip) and require additional H(4)Bip for maximal activity. We examined the effects of H(4)Bip and pterin -derived inhibitors (antipterins) on purified neuronal NOS-I quaternary str ucture and H(4)Bip content. During L-arginine turnover, NOS-I dimers time d ependently dissociated into inactive monomers, paralleled by a loss of enzy me-associated pterin. Dimer dissociation was inhibited when saturating leve ls of H(4)Bip were added during catalysis, Similar results were obtained wi th pterin-free NOS-I expressed in Escherichia coli. This stabilizing effect of H(4)Bip was mimicked by the anti-pterin 2-amino-4,6-dioxo-3,4,5,6,8,8a, 9, 10-octahydro-oxazolo [1,2f]-pteridine (PHS-32), which also displaced NOS -associated H(4)Bip in a competitive manner. Surprisingly, H(4)Bip not only dissociated from NOS during catalysis, but was only partially recovered in the solute (50.0 +/- 16.5% of control at 20 min). NOS-associated H(4)Bip a ppeared to react with a NOS catalysis product to a derivative distinct from dihydrobiopterin or biopterin. Under identical conditions, reagent H(4)Bip was chemically stable and fully recovered (95.5 +/- 3.4% of control). A si milar loss of both reagent and enzyme-bound H(4)Bip and dimer content was o bserved by NO generated from spermine NONO-ate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during cata lysis, possibly by interfering with enzyme destabilizing products.