Formation of N delta-cyanoornithine from NG-hydroxy-L-arginine and hydrogen peroxide by neuronal nitric oxide synthase: implications for mechanism.
Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation of NG-hydroxy-L-arginine (NHA) by hydrogen peroxide. The amino acid products were characterized by high-performance liquid chromatography/mass spectrometry of the o-phthalaldehyde/2-mercaptoethanol derivatives and identified as citrulline and N delta-cyanoornithine (CN-orn). The assignment of CN-orn was confirmed by independent chemical synthesis and comparison of the properties of the enzyme-derived product with those of synthetic CN-orn. The inorganic products detected in the enzymatic reaction were NO2- and NO3-, presumably from oxidation of NO-. The reaction of H2O2 and NHA with nNOS was at least 10-fold slower than the reaction of NADPH, O2, and NHA (Vmax,app = 49 +/- 2 nmol min-1 mg-1 for the reactions with 10 microM added H4B). The reaction exhibited saturation kinetics with respect to hydrogen peroxide [K(m,app)(H2O2) = 10 +/- 1 mM for the reactions with 10 microM added H4B]. No H2O2-dependent reaction was observed with L-arginine as the amino acid substrate. The different products for the NADPH- and H2O2-dependent transformations of NHA are of mechanistic significance in the NOS reaction.
Clague
M J
MJ
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Wishnok
J S
JS
Marletta
M A
MA
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008956:Models, Chemical; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009952:Ornithine / Q000031:analogs & derivatives / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D013379:Substrate Specificity; D014162:Transfection
D011994:Recombinant Proteins; D002956:Citrulline; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D006861:Hydrogen Peroxide; D009952:Ornithine; D019001:Nitric Oxide Synthase