Oxygenase Domain of Drosophila melanogaster Nitric Oxide Synthase: Unique Kinetic Parameters Enable a More Efficient NO Release

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• 作者：Sougata Sinha Ray ; Jesú ; s Tejero ; Zhi-Qiang Wang ; Tanmay Dutta ; Arindam Bhattacharjee ; Michael Regulski ; Tim Tully ; Sanjay Ghosh ; Dennis J. Stuehr
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 23, 2007
• 年：2007
• 卷：46
• 期：42
• 页码：11857 - 11864
• 全文大小：149K
• 年卷期：v.46,no.42(October 23, 2007)
• ISSN：1520-4995

文摘

Although nitric oxide (NO) is important for cell signaling and nonspecific immunity in thefruit fly Drosophila melanogaster, little is known about its single NO synthase (dNOS). We expressedthe oxygenase domain of dNOS (dNOSoxy), characterized its spectroscopic, kinetic, and catalytic properties,and interpreted them in light of a global kinetic model for NO synthesis. Single turnover reactions withferrous dNOSoxy showed it could convert Arg to N'-hydroxy-L-arginine (NOHA), or NOHA to citrullineand NO, when it was given 6R-tetrahydrobiopterin and O₂. The dNOSoxy catalyzed Arg hydroxylationand NOHA oxidation at rates that matched or exceeded the rates catalyzed by the three mammalian NOSoxyenzymes. Consecutive heme-dioxy, ferric heme-NO, and ferric heme species were observed in the NOHAreaction of dNOSoxy, indicating that its catalytic mechanism is the same as in the mammalian NOS.However, NO dissociation from dNOSoxy was 4 to 9 times faster than that from the mammalian NOSenzymes. In contrast, the dNOSoxy ferrous heme-NO complex was relatively unreactive toward O₂ andin this way was equivalent to the mammalian neuronal NOS. Our data show that dNOSoxy has uniquesettings for the kinetic parameters that determine its NO synthesis. Computer simulations reveal thatthese unique settings should enable dNOS to be a more efficient and active NO synthase than themammalian NOS enzymes, which may allow it to function more broadly in cell signaling and immunefunctions in the fruit fly.