title: Tetrahydrobiopterin inhibits monomerization and is consumed during catalysis in neuronal NO synthase.
abstract: The biosynthesis of nitric oxide (NO) is catalyzed by homodimeric NO synthases (NOS). For unknown reasons, all NOS co-purify with substoichiometric amounts 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 (anti-pterins) on purified neuronal NOS-I quaternary structure and H(4)Bip content. During L-arginine turnover, NOS-I dimers time dependently dissociated into inactive monomers, paralleled by a loss of enzyme-associated pterin. Dimer dissociation was inhibited when saturating levels of H(4)Bip were added during catalysis. Similar results were obtained with 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 appeared 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 similar loss of both reagent and enzyme-bound H(4)Bip and dimer content was observed by NO generated from spermine NONOate. In conclusion, we propose a role for H(4)Bip as a dimer-stabilizing factor of neuronal NOS during catalysis, possibly by interfering with enzyme destabilizing products.
authors.affiliation: Department of Pharmacology and Toxicology, Julius-Maximilians-University, Würzburg, 97078 Germany. a.reif@gmx.net
authors.forename: A
authors.initials: A
authors.lastname: Reif
authors.forename: L G
authors.initials: LG
authors.lastname: Fröhlich
authors.forename: P
authors.initials: P
authors.lastname: Kotsonis
authors.forename: A
authors.initials: A
authors.lastname: Frey
authors.forename: H M
authors.initials: HM
authors.lastname: Bömmel
authors.forename: D A
authors.initials: DA
authors.lastname: Wink
authors.forename: W
authors.initials: W
authors.lastname: Pfleiderer
authors.forename: H H
authors.initials: HH
authors.lastname: Schmidt
mesh_terms: D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D019281:Dimerization; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D011487:Protein Conformation; D011621:Pteridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry; D013053:Spectrophotometry; D013096:Spermine / Q000031:analogs & derivatives
chemical_list: D011621:Pteridines; D011994:Recombinant Proteins; D001708:Biopterins; D013096:Spermine; D009569:Nitric Oxide; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C003402:sapropterin