NITROARGININE AND TETRAHYDROBIOPTERIN BINDING TO THE HEME 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 ox ygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-L-biopterin (tetrah ydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A nove l scintillation proximity assay has been established for equilibrium a nd kinetic measurements of substrate, inhibitor and cofactor binding t o a recombinant N-terminal haem-binding domain of rat neuronal NOS (nN OS). 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 di ssociation 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 val ues 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 inhibitor s, L-N-G-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornith ine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [H-3]Tetrahydrobiopterin bound to the nNOS haem domain w ith an apparent K-d of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction wit h tetrahydrobiopterin were complex, showing a triphasic binding proces s and a single off rate. An alternating catalytic site mechanism for N OS is proposed.