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, k2, 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...