Two modes of regulation mechanism in the successive reaction of rat neuronal nitric oxide synthase

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The conversion of l-arginine (Arg) to N ω-hydroxy- l-arginine (OH-Arg) and further to l-citrulline (Cit) in one cycle of the reaction of purified rat neuronal nitric oxide synthase (nNOS) was measured with the reaction rapid-quenching method using 3H-Arg as the substrate. The produced 3H-OH-Arg was successively converted into 3H-Cit without leaving enzyme. From analysis of the time courses, it was found that the rate-determining step of the multi-cycle reaction of nNOS in the presence of excess amount of Arg was dissociation of produced 3H-Cit from nNOS. With decrease in NADPH concentration, the ratio of amounts of produced OH-Arg/Cit increased under the multi-cycle reaction condition. Under the single cycle reaction condition, decrease in NADPH concentration caused decrease in the rate of hydroxylation reactions both for Arg and OH-Arg but hardly affected the dissociation rate of OH-Arg from the nNOS. On the other hand, decrease in CaM concentration did not change the hydroxylation rate from Arg to OH-Arg but decreased the amount of active complex (CaM–nNOS) under the single cycle reaction condition. Under multi-cycle reaction condition, decrease in CaM concentration did not affect the ratio of produced OH-Arg/Cit. It became clear that there are two modes of regulation in the nNOS reaction.