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 (kcat1 and kcat2) are both limited by the rate of ferric heme reduction (kr). An enzyme ferric heme-NO complex forms as an immediate product complex and then undergoes either dissociation (at a rate that we denote as kd) to release NO in a productive manner, or reduction (kr) 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 (kcat1, k cat2, kr, kd and kox) 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. Global kinetic model for NOS catalysis. NO Synthase enzymes convert L-arginine to NO in two sequential reactions. During NOS catalysis five key kinetic parameters (kcat1, kcat2, kr, kd, and kox) play major role. The interplay of these five kinetic parameters determine NOS specific activity, O2 concentration response, and pulsatile versus steady-state NO generation. © 2013 FEBS.