L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen.
Previously proposed mechanisms for the production of L-citrulline from L-arginine by macrophage nitric oxide (NO.) synthase involve either hydrolysis of arginine or hydration of an intermediate and thus predict incorporation of water oxygen into L-citrulline. Macrophage NO. synthase was incubated with L-arginine, NADPH, tetrahydrobiopterin, FAD, and dithiothreitol in H2(18)/16O2. L-Citrulline produced in this reaction was analyzed with gas chromatography/mass spectrometry. Its mass spectrum matched that of L-citrulline generated in H2(16)O/16O2. The base fragment ion of m/z 99 was shown to contain the ureido carbonyl group by using L-[guanidino-13C]arginine as substrate. When the enzyme reaction was performed in H2(16)O/18O2, the base fragment ion shifted to m/z 101 with L-[guanidino-12C]arginine as the substrate and to m/z 102 with L-[guanidino-13C]arginine. These results indicate that the ureido oxygen of the L-citrulline product of macrophage NO.synthase derives from dioxygen and not from water.
Beatrice and Samuel A. Seaver Laboratory, Department of Medicine, Cornell University Medical College, New York, New York 10021.
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Kwon
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Nathan
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Gilker
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Griffith
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Matthews
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Stuehr
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D002956:Citrulline / Q000096:biosynthesis*; D003600:Cytosol / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology*; D013058:Mass Spectrometry; D019001:Nitric Oxide Synthase; D010100:Oxygen / Q000378:metabolism*; D010103:Oxygen Isotopes
D008070:Lipopolysaccharides; D010103:Oxygen Isotopes; D002956:Citrulline; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D010100:Oxygen