Kinetic studies on the successive reaction of neuronal nitric oxide synthase from L-arginine to nitric oxide and L-citrulline

Rat neuronal nitric oxide synthase (nNOS) was heterologously expressed in E scherichia coli and purified. The conversion of L-arginine to N-omega-hydro xy-L-arginine and further to L-citrulline in one cycle of the reaction of t he purified nNOS was measured with the reaction rapid quenching method usin g H-3-L-arginine as the substrate. It was found that most of the produced H -3-N-omega-hydroxy-L-arginine was successively hydroxylated to H-3-L-citrul line without leaving the enzyme. From the analysis of time courses, the rat e constants for each reaction step, and also for the dissociation of the in termediate, were estimated at various temperature in which the rates for th e first and the second reactions were not much different each other but the rate for the dissociation of H-3-N-omega-hydroxy-L-arginine from the enzym e was significantly slow. Under the steady-state reaction condition, almost all of the nNOS was estimated to be active from the amount of burst format ion of L-citrulline in the pre-steady state. The rate constant for the diss ociation of the product L-citrulline from nNOS was calculated from the comb ination of results of the rapid quenching experiments and the metabolism of L-arginine in the presence of an excess amount of substrate, which was the smallest among all the rate constants in one cycle of the nNOS reaction. T he activation energies for all the reaction steps were determined from the temperature dependence of the rate constants, which revealed that the rate- determining step of the nNOS reaction in the steady state was the dissociat ion of the product L-citrulline from the enzyme.