Neuronal nitric oxide synthase: substrate and solvent kinetic isotope effects on the steady-state kinetic parameters for the reduction of 2,6-dichloroindophenol and cytochrome c(3+).
The neuronal nitric oxide synthase (nNOS) basal and calmodulin- (CaM-) stimulated reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) follow ping-pong mechanisms [Wolthers and Schimerlik (2001) Biochemistry 40, 4722-4737]. Primary deuterium [NADPH(D)] and solvent deuterium isotope effects on the kinetic parameters were studied to determine rate-limiting step(s) in the kinetic mechanisms for the two substrates. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Values for (D)V and (D)(V/K)(NADPH) were similar for the basal (1.3-1.7) and CaM-stimulated (1.5-2.1) reduction of DCIP, while (D)V (2.1-2.8) was higher than (D)(V/K)(NADPH) (1.1-1.5) for cytochrome c(3+) reduction with and without CaM. This suggests that the rate of the reductive half-reaction (NADPH oxidation) rather than that of the oxidative half-reaction (reduction of DCIP or cytochrome c(3+)) limits the overall reaction rate. A value for (D)(V/K)(NADPH) close to 1 indicates the intrinsic isotope effect on hydride transfer is suppressed by a slower step in the reductive half-reaction. The oxidative half-reaction is insensitive to NADPD isotope effects as both (D)(V/K)(DCIP) and (D)(V/K)(cytc) equal 1 within experimental error. Large solvent kinetic isotope effects (SKIE) observed for (V/K)(cytc) for basal (approximately 8) and CaM-stimulated (approximately 31) reduction of cytochrome c(3+) suggest that proton uptake from the solvent limits the rate of the oxidative half-reaction. This step does not severely limit the overall reaction rate as (D2O)V equaled 2 and (D2O)(V/K)(NADPH) was between 0.9 and 1.3 for basal and CaM-stimulated cytochrome c(3+) reduction.
Wolthers
Kirsten R
KR
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Schimerlik
Michael I
MI
D015086:2,6-Dichloroindophenol / Q000737:chemistry*; D000818:Animals; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004579:Electron Transport; D007202:Indicators and Reagents / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D003574:Cytochrome c Group; D007202:Indicators and Reagents; D009249:NADP; D015086:2,6-Dichloroindophenol; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat