Nitroarginine and tetrahydrobiopterin binding to the haem domain of neuronal nitric oxide synthase using a scintillation proximity assay

Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent K-d values for nNOS haem-domain-binding of arginine and N-omega-nitro-L-arginine (nitroarginine) were measured as 1.6 mu M and 25 nM respectively. The kinetics of [H-3]nitroarginine binding and dissociation yielded an association rate constant of 1.3 x 10(4) s(-1) . M-1 and a dissociation rate constant of 1.2 x 10(-4) s(-1). These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent K-d values were compared and were found to be similar for the inhibitors, L-N-G-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [H-3]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent K-d of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.