Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase.
The kinetics of binding L-arginine and three alternative substrates (homoarginine, N-methylarginine, and N-hydroxyarginine) to neuronal nitric oxide synthase (nNOS) were characterized by conventional and stopped-flow spectroscopy. Because binding these substrates has only a small effect on the light absorbance spectrum of tetrahydrobiopterin-saturated nNOS, their binding was monitored by following displacement of imidazole, which displays a significant change in Soret absorbance from 427 to 398 nm. Rates of spectral change upon mixing Im-nNOS with increasing amounts of substrates were obtained and found to be monophasic in all cases. For each substrate, a plot of the apparent rate versus substrate concentration showed saturation at the higher concentrations. K(-)(1), k(2), k(-)(2), and the apparent dissociation constant were derived for each substrate from the kinetic data. The dissociation constants mostly agreed with those calculated from equilibrium spectral data obtained by titrating Im-nNOS with each substrate. We conclude that nNOS follows a two-step, reversible mechanism of substrate binding in which there is a rapid equilibrium between Im-nNOS and the substrate S followed by a slower isomerization process to generate nNOS'-S: Im-nNOS + S if Im-nNOS-S if nNOS'-S + Im. All four substrates followed this general mechanism, but differences in their kinetic values were significant and may contribute to their varying capacities to support NO synthesis.
Abu-Soud
H M
HM
Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Wang
J
J
Rousseau
D L
DL
Stuehr
D J
DJ
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002384:Catalysis; D002478:Cells, Cultured; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D046911:Macromolecular Substances; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D007093:Imidazoles; D046911:Macromolecular Substances; D009569:Nitric Oxide; C029899:imidazole; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat