Interaction between Neuronal Nitric-Oxide Synthase and Tetrahydrobiopterin Revisited: Studies on the Nature and Mechanism of Tight Pterin Binding

详细信息    查看全文

• 作者：Christian L. Heine ; Bernd Kolesnik ; Renate Schmidt ; Ernst R. Werner ; Bernd Mayer ; Antonius C. F. Gorren
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：March 4, 2014
• 年：2014
• 卷：53
• 期：8
• 页码：1284-1295
• 全文大小：509K
• 年卷期：v.53,no.8(March 4, 2014)
• ISSN：1520-4995

文摘

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^鈥?鈥?0^鈥? 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 鈮?2 脳 10^鈥? 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 渭M BH4 (2 min with 1 渭M 4-amino-BH4) at 4 掳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.