Multiple catalytic functions of brain nitric oxide synthase. Biochemical characterization, cofactor-requirement, and the role of N omega-hydroxy-L-arginine as an intermediate.
Brain NO (nitric oxide) synthase contains FAD, FMN, heme, and tetrahydrobiopterin as prosthetic groups and represents a multi-functional oxidoreductase catalyzing oxidation of L-arginine to NO and L-citrulline, formation of H2O2, and reduction of cytochrome c. We show that substrate analogues and inhibitors interacting with the heme block both the reductive activation of oxygen and the oxidation of L-arginine without affecting cytochrome c reduction. We further demonstrate that N omega-hydroxy-L-arginine is an intermediate in enzymatic NO synthesis. The ratio of L-citrulline to free N omega-hydroxy-L-arginine was > or = 50 under various assay conditions, but could markedly be reduced down to 4 by redox active inhibitors. Brain NO synthase is shown to utilize both L-arginine and N omega-hydroxy-L-arginine for the formation of stoichiometric amounts of NO and L-citrulline. Tetrahydrobiopterin equally enhanced reaction rates from either substrate (approximately 5-fold), but its rate accelerating effects were only observed at NADPH concentrations > or = 3 microM. In the absence of L-arginine or tetrahydrobiopterin, brain NO synthase catalyzes the generation of H2O2. We now show that, in contrast to L-arginine, N omega-hydroxy-L-arginine fully blocked H2O2 formation in the absence of exogenous tetrahydrobiopterin, indicating that N omega-hydroxy-L-arginine is a direct inhibitor of enzymatic oxygen activation. Based on these data, a hypothetical mechanism of enzymatic NO formation is discussed.
Klatt
P
P
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitatsplatz, Austria.
Schmidt
K
K
Uray
G
G
Mayer
B
B
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D003067:Coenzymes / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D013552:Swine
D003067:Coenzymes; D001708:Biopterins; D006418:Heme; D009249:NADP; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; C003402:sapropterin