Competition and binding of arginine, imidazole, and aminoguanidine to endothelial nitric oxide synthase: aminoguanidine is a poor model for substrate, intermediate, and arginine analog inhibitor binding.
The oxygenase domains of nitric oxide synthases are unusual in that they contain at least three ligand binding sites; these correspond to the axial heme ligand position, the substrate binding site, and the pterin binding site. Ligands can occupy portions of a site or extend into regions of adjacent sites. Depending on the size, shape, and binding mode of ligands to these positions, cooperative and anticooperative interactions mediated conformationally and by binding domain overlap can be observed. In the present study we describe competition between arginine and imidazole at the axial heme ligand position; a second imidazole, which occupies part of the arginine site in some crystal structures, is too weak to contribute to the equilibria. All spectroscopic titrations using imidazole competition depend on displacement of the heme axial imidazole ligand, which drives the ferriheme low spin. Aminoguanidine, a partial arginine analog, has multiple binding modes. It is somewhat competitive with arginine; a ternary complex forms, but the K(d) for arginine increases from 1 to 15 microM in the presence of saturating aminoguanidine. Aminoguanidine competition with imidazole is very weak, amounting to approximately a factor of two increase in K(d). This implies that aminoguanidine has multiple binding modes and is not well described as an arginine analog. The major binding mode occupies part of the binding site but does not extend into the imidazole axial ligand binding domain and probably corresponds to the crystal structure. The other binding mode is not significantly overlapped with the arginine site.
Katsumoto
Satoshi
S
Biology Department, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, USA.
Smith
Susan M E
SM
Martasek
Pavel
P
Salerno
John C
JC
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001667:Binding, Competitive; D002417:Cattle; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006146:Guanidines / Q000378:metabolism / Q000494:pharmacology; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding; D013057:Spectrum Analysis; D013379:Substrate Specificity; D017186:Titrimetry
D004791:Enzyme Inhibitors; D006146:Guanidines; D007093:Imidazoles; C029899:imidazole; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C004479:pimagedine