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 Kd values for nNOS haem-domain-binding of arginine and Nomega-nitro-L-arginine (nitroarginine) were measured as 1.6 microM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3x10(4) s-1.M-1 and a dissociation rate constant of 1.2x10(-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 Kd values were compared and were found to be similar for the inhibitors, L-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-L-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd 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.
Alderton
W K
WK
Glaxo Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts. SG1 2NY, UK. wka34454@glaxowellcome.co.uk
Boyhan
A
A
Lowe
P N
PN
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D001667:Binding, Competitive; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006418:Heme / Q000378:metabolism*; D007191:Indazoles / Q000494:pharmacology; D007550:Isothiuronium / Q000031:analogs & derivatives / Q000494:pharmacology; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D019335:Nitroarginine / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry; D010105:Oxygenases / Q000737:chemistry; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D004791:Enzyme Inhibitors; D007191:Indazoles; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D019335:Nitroarginine; D001708:Biopterins; D007550:Isothiuronium; C007687:etiron; D006418:Heme; D001120:Arginine; D010088:Oxidoreductases; D010105:Oxygenases; D019001:Nitric Oxide Synthase; C003402:sapropterin; C080122:7-nitroindazole