Stopped-flow analysis of substrate binding to neuronal nitric oxide synthase

Husam M. Abu-Soud, Jianling Wang, Denis L. Rousseau, Dennis J. Stuehr

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


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. 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 mutually implies Im-nNOS - S mutually implies 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.

Original languageEnglish (US)
Pages (from-to)12446-12451
Number of pages6
Issue number38
StatePublished - Sep 21 1999

ASJC Scopus subject areas

  • Biochemistry


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