Characteristics of the nitric oxide synthase-catalyzed conversion of arginine to N-hydroxyarginine, the first oxygenation step in the enzymic synthesis of nitric oxide.
The nitric oxide synthase-catalyzed conversion of L-arginine to L-citrulline and nitric oxide is known to be the sum of two partial reactions: oxygenation of arginine to N-hydroxyarginine, followed by oxygenation of N-hydroxyarginine to citrulline and nitric oxide. Whereas the conversion of N-hydroxyarginine to citrulline and nitric oxide has been the subject of a number of studies, the oxygenation of arginine to N-hydroxyarginine has received little attention. Here we show that substrate amounts of rat cerebellar nitric oxide synthase, in the absence of added NADPH, catalyze the conversion of arginine to N-hydroxyarginine as the dominant product. The product appears not to be tightly bound to the enzyme. A maximum of 0.16 mol of N-hydroxyarginine/mol of nitric oxide synthase subunit was formed. The reaction requires oxygen and the addition of Ca2+/calmodulin and is stimulated 3-fold by tetrahydrobiopterin. Upon addition of NADPH, citrulline is formed exclusively. Conversion of N-hydroxyarginine to citrulline, like the first partial reaction, requires Ca2+/calmodulin and is stimulated by tetrahydrobiopterin but differs from the first partial reaction in being completely dependent upon addition of NADPH. These results indicate that brain nitric oxide synthase contains an endogenous reductant that can support oxygenation of arginine but not of N-hydroxyarginine. The reductant is not NADPH, since the amount of nitric oxide synthase-bound NADPH is appreciably less than the amount required for N-hydroxyarginine synthesis. Possible candidates for this role are discussed in relation to proposed mechanisms of action of nitric oxide synthase.
Campos
K L
KL
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20895.
Giovanelli
J
J
Kaufman
S
S
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism; D006801:Humans; D007668:Kidney; D007700:Kinetics; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection; D014316:Tritium
D011994:Recombinant Proteins; D014316:Tritium; D002956:Citrulline; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases