Reaction of neuronal nitric-oxide synthase with 2,6-dichloroindolphenol and cytochrome c3+: influence of the electron acceptor and binding of Ca2+-activated calmodulin on the kinetic mechanism.
Binding of Ca(2+)-activated calmodulin (Ca(2+)-CaM) to neuronal nitric-oxide synthase (nNOS) increases the rate of 2,6-dichloroindolphenol (DCIP) reduction 2-3-fold and that of cytochrome c(3+) 10-20-fold. Parallel initial velocity patterns indicated that both substrates were reduced via two-half reactions in a ping-pong mechanism. Product and dead-end inhibition data with DCIP were consistent with an iso ping-pong bi-bi mechanism; however, product and dead-end inhibition studies with cytochrome c(3+) were consistent with the (two-site) ping-pong mechanism previously described for the NADPH-cytochrome P450 reductase-catalyzed reduction of cytochrome c(3+) [Sem, D., and Kasper, C. (1994) Biochemistry 33, 12012--12021]. Dead-end inhibition by 2'-adenosine monophosphate (2'AMP) was competitive versus NADPH for both electron acceptors, although the value of the slope inhibition constant, K(is), was 25-30-fold greater with DCIP as the substrate than with cytochrome c(3+). The difference in the apparent affinity of 2'AMP is proposed to result from a rapidly equilibrating isomerization step that occurs in both mechanisms prior to the binding of NADPH. Thus, initial velocity, product, and dead-end inhibition data were consistent with a di-iso ping-pong bi-bi and an iso (two-site) ping-pong mechanism for the reduction of DCIP and cytochrome c(3+), respectively. The presence Ca(2+)-CaM did not alter the proposed kinetic mechanisms. The activated cofactor had a negligible effect on (k(cat)/K(m))(NADPH), while it increased (k(cat)/K(m))(DCIP) and (k(cat)/K(m))(cytc) 4.5- and 23-fold, respectively.
Wolthers
K R
KR
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.
Schimerlik
M I
MI
D015086:2,6-Dichloroindophenol / Q000737:chemistry*; D000249:Adenosine Monophosphate / Q000737:chemistry; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003574:Cytochrome c Group / Q000037:antagonists & inhibitors / Q000737:chemistry*; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000737:chemistry; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D011485:Protein Binding; D051381:Rats; D013379:Substrate Specificity
D002147:Calmodulin; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D000249:Adenosine Monophosphate; D009249:NADP; C017165:cytochrome c(3); D015086:2,6-Dichloroindophenol; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; D002118:Calcium