title: Thermodynamic characterization of five key kinetic parameters that define neuronal nitric oxide synthase catalysis.
abstract: NO synthase (NOS) enzymes convert L-arginine to NO in two sequential reactions whose rates (k(cat1) and k(cat2)) are both limited by the rate of ferric heme reduction (k(r)). An enzyme ferric heme-NO complex forms as an immediate product complex and then undergoes either dissociation (at a rate that we denote as k(d)) to release NO in a productive manner, or reduction (k(r)) to form a ferrous heme-NO complex that must react with O2 (at a rate that we denote as k(ox)) in a NO dioxygenase reaction that regenerates the ferric enzyme. The interplay of these five kinetic parameters (k(cat1), k(cat2), k(r), k(d) and k(ox)) determines NOS specific activity, O2 concentration response, and pulsatile versus steady-state NO generation. In the present study, we utilized stopped-flow spectroscopy and single catalytic turnover methods to characterize the individual temperature dependencies of the five kinetic parameters of rat neuronal NOS. We then incorporated the measured kinetic values into computer simulations of the neuronal NOS reaction using a global kinetic model to comprehensively model its temperature-dependent catalytic behaviours. The results obtained provide new mechanistic insights and also reveal that the different temperature dependencies of the five kinetic parameters significantly alter neuronal NOS catalytic behaviours and NO release efficiency as a function of temperature.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, OH 44195, USA.
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.forename: Jesús
authors.initials: J
authors.lastname: Tejero
authors.forename: Mekki
authors.initials: M
authors.lastname: Bayachou
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Mohammed
authors.initials: M
authors.lastname: Fadlalla
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D055162:Biocatalysis; D003198:Computer Simulation; D005296:Ferrous Compounds / Q000737:chemistry / Q000378:metabolism; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000201:enzymology; D009569:Nitric Oxide / Q000096:biosynthesis / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
chemical_list: D005296:Ferrous Compounds; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D001120:Arginine; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D010100:Oxygen
keywords: electron transfer; heme protein; heme reduction; nitric oxide synthase; stopped-flow