title: Interaction between neuronal nitric-oxide synthase and tetrahydrobiopterin revisited: studies on the nature and mechanism of tight pterin binding.
abstract: Recombinant neuronal nitric-oxide synthase (nNOS) expressed in baculovirus-infected Sf9 cells contains approximately 1 equiv of tightly bound tetrahydrobiopterin (BH4) per dimer and binds a second equivalent with a dissociation constant in the 10(-7)-10(-6) M range. Less is known about the pterin-binding properties of nNOS originating from expression systems such as Escherichia coli that do not produce BH4. We determined the binding properties of E. coli-expressed nNOS for BH4 and several inhibitory pterins by monitoring their effects on enzyme activity. E. coli-expressed nNOS as isolated was activated by BH4 monophasically with EC50 ≈ 2 × 10(-7) M, demonstrating a lack of tight pterin binding. However, overnight incubation with BH4 resulted in tight binding of one BH4 per dimer, yielding an enzyme that resembled Sf9-expressed nNOS. Tight pterin binding was also induced by preincubation with 4-amino-tetrahydrobiopterin, but not by 7,8-dihydrobiopterin or 4-amino-dihydrobiopterin, suggesting that tight-binding site formation requires preincubation with a fully reduced pteridine. Kinetic experiments showed that tight-binding site formation takes approximately 10 min with 1 μM BH4 (2 min with 1 μM 4-amino-BH4) at 4 °C. Anaerobic preincubation experiments demonstrated that O2 is not involved in the process. Gel electrophoretic studies suggest that tight-binding site formation is accompanied by an increase in the strength of the NOS dimer. We propose that incubation of pterin-free nNOS with BH4 creates one tight pterin-binding site per dimer, leaving the other site unaffected, in a reaction that involves redox chemistry.
authors: Department of Pharmacology and Toxicology, Institute of Pharmaceutical Sciences, Karl-Franzens-University Graz , A-8010, Graz, Austria.
authors: Christian L
authors: CL
authors: Heine
authors: Bernd
authors: B
authors: Kolesnik
authors: Renate
authors: R
authors: Schmidt
authors: Ernst R
authors: ER
authors: Werner
authors: Bernd
authors: B
authors: Mayer
authors: Antonius C F
authors: AC
authors: Gorren
mesh_terms: D000818:Animals; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D004795:Enzyme Stability; D007700:Kinetics; D009569:Nitric Oxide / Q000096:biosynthesis; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D051381:Rats; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: C106930:4-amino-tetrahydrobiopterin; D001708:Biopterins; D009569:Nitric Oxide; C017226:7,8-dihydrobiopterin; D052248:Nitric Oxide Synthase Type I; C003402:sapropterin; D010100:Oxygen