title: A kinetic simulation model that describes catalysis and regulation in nitric-oxide synthase.
abstract: After initiating NO synthesis a majority of neuronal NO synthase (nNOS) quickly partitions into a ferrous heme-NO complex. This down-regulates activity and increases enzyme K(m,O(2)). To understand this process, we developed a 10-step kinetic model in which the ferric heme-NO enzyme forms as the immediate product of catalysis, and then partitions between NO dissociation versus reduction to a ferrous heme-NO complex. Rate constants used for the model were derived from recent literature or were determined here. Computer simulations of the model precisely described both pre-steady and steady-state features of nNOS catalysis, including NADPH consumption and NO production, buildup of a heme-NO complex, changes between pre-steady and steady-state rates, and the change in enzyme K(m,O(2)) in the presence or absence of NO synthesis. The model also correctly simulated the catalytic features of nNOS mutants W409F and W409Y, which are hyperactive and display less heme-NO complex formation in the steady state. Model simulations showed how the rate of heme reduction influences several features of nNOS catalysis, including populations of NO-bound versus NO-free enzyme in the steady state and the rate of NO synthesis. The simulation predicts that there is an optimum rate of heme reduction that is close to the measured rate in nNOS. Ratio between NADPH consumption and NO synthesis is also predicted to increase with faster heme reduction. Our kinetic model is an accurate and versatile tool for understanding catalytic behavior and will provide new perspectives on NOS regulation.
authors.affiliation: Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: J
authors.initials: J
authors.lastname: Santolini
authors.forename: S
authors.initials: S
authors.lastname: Adak
authors.forename: C M
authors.initials: CM
authors.lastname: Curran
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D002956:Citrulline / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D002956:Citrulline; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I