Binding of L-arginine and imidazole suggests heterogeneity of rat brain neuronal nitric oxide synthase.
Nitric oxide synthase (NOS) is inhibited by imidazole, which binds to the heme in a low-spin complex absorbing at 428 nm. Conversion by L-arginine of this complex into a high-spin species absorbing at 395 nm is a common method to determine the binding parameters of Arg. However, both Arg-competitive and noncompetitive inhibition of NOS by imidazole has been reported, and optical studies with neuronal NOS provided no evidence for imidazole affecting Arg binding. We investigated the cause for these paradoxical observations with recombinant rat brain neuronal NOS. Imidazole bound to nNOS with a K(d)(app) of 50 microM; tetrahydrobiopterin (BH4) lowered the affinity of nNOS for imidazole 4-fold. The enzyme behaved heterogeneously with respect to Arg binding. Most of nNOS (65-80%) showed competition between Arg and imidazole. In the presence of BH4, a K(d)(Arg) of 1 microM could be estimated for this fraction, as well as apparent association and dissociation rate constants of 2.5 x 10(6) M(-1) x s(-1) and 2.5 s(-1). A second fraction of nNOS (20-30%) exhibited little or no competition. Consequently, Arg binding did not cause dissociation of the imidazole complex for this fraction, and complete generation of the high-spin state by Arg could not be achieved in the presence of imidazole. A third fraction (< or =10%) bound Arg with low affinity (K(d) 1-2 mM). Because of this heterogeneity, titration curves with Arg became almost uninterpretable. We propose that this heterogeneous response of nNOS toward Arg and imidazole is underlying the apparently conflicting results reported in the literature.
Gorren
Antonius C F
AC
Institut für Pharmakologie und Toxikologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria. antonius.gorren@kfunigraz.ac.at
Schmidt
Kurt
K
Mayer
Bernd
B
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive / Q000187:drug effects; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D006418:Heme / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D018411:Spodoptera; D017186:Titrimetry; D014407:Tumor Cells, Cultured
D007093:Imidazoles; D009419:Nerve Tissue Proteins; D001708:Biopterins; D006418:Heme; C029899:imidazole; D001120:Arginine; C496320:NOS1 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse; C496322:Nos1 protein, rat; C003402:sapropterin