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 c3+
| Autor: | Kirsten R. Wolthers, Michael I. Schimerlik |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Inorganic chemistry
Cytochrome c Group Nitric Oxide Synthase Type I Oxidative phosphorylation Binding Competitive Biochemistry Medicinal chemistry Catalysis Substrate Specificity Electron Transport Calmodulin Kinetic isotope effect Animals biology Chemistry Cytochrome c NADPH oxidation Electron transport chain Rats Nitric oxide synthase Kinetics Models Chemical Deuterium biology.protein 2 6-Dichloroindophenol Indicators and Reagents Steady state (chemistry) Nitric Oxide Synthase Oxidation-Reduction NADP Protein Binding |
| Zdroj: | Biochemistry. 41:196-204 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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