Inhibitors of brain nitric oxide synthase. Binding kinetics, metabolism, and enzyme inactivation.
Nitric oxide (NO) is synthesized from L-arginine by different NO synthase isozymes, which are inhibited by the substrate analogs NG-methyl- and NG-nitro-L-arginine. We studied binding of 3H-labeled NG-nitro-L-arginine to purified brain NO synthase and compared the data with results obtained in enzyme kinetic experiments. Binding data revealed a single binding site for NG-nitro-L-[3H]arginine (KD = 0.17 microM). Binding was competitively antagonized by L-arginine (KI = 2.9 microM). The half-time of dissociation was remarkably slow (9.4 min) and closely correlated with the time necessary for surmounting NO synthase inhibition by dilution. Although an apparently less potent inhibitor, NG-methyl-L-arginine exhibited the same affinity for brain NO synthase as the nitro derivative (KI = 0.17 microM), and in initial rate experiments, almost equal KI values were obtained for NG-methyl-L-arginine (0.61 microM) and NG-nitro-L-arginine (0.53 microM). However, after prolonged incubation periods, NG-nitro-L-arginine induced a rapid inactivation of the enzyme, whereas the methyl derivative turned out to be a substrate of NO synthase, which was slowly converted into stoichiometric amounts of NO and L-citrulline.
Klatt
P
P
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Austria.
Schmidt
K
K
Brunner
F
F
Mayer
B
B
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D001921:Brain / Q000201:enzymology*; D002850:Chromatography, Gel; D007700:Kinetics; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D012044:Regression Analysis; D013552:Swine; D019323:omega-N-Methylarginine
D019335:Nitroarginine; D019323:omega-N-Methylarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases