Interaction between Neuronal Nitric-Oxide Synthaseand Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanismof Tight Pterin Binding

摘要

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 experimentsshowed that tight-binding site formation takes approximately 10 minwith 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 suggestthat tight-binding site formation is accompanied by an increase inthe strength of the NOS dimer. We propose that incubation of pterin-freenNOS with BH4 creates one tight pterin-binding site per dimer, leavingthe other site unaffected, in a reaction that involves redox chemistry.