Cyanide binding study of neuronal nitric oxide synthase: effects of inhibitors and mutations at the substrate binding site.
In order to understand the heme distal structure of neuronal nitric oxide synthase (nNOS), we studied cyanide binding to the ferric wild-type and substrate binding site mutants, Glu592Ala and Tyr588His, of the isolated oxygenase domain in the absence and presence of substrates and inhibitors. Cyanide bound to isolated heme-bound oxygenase domains (nNOSox) in the absence of the substrates with the dissociation constant (K(d)) of 3.1 mM. The presence of the substrates, L-Arg and NHA, did not change the K(d) value. However, cyanide binding was almost abolished in the presence of inhibitors such as NAME, thiocitrulline and 7-NI. The effect of the inhibitors were not observed for the Glu592Ala mutant, while similar strong inhibiting effects were observed for the Tyr588His mutant. We discuss the binding fashion of those inhibitors to the heme substrate binding site of nNOS.
Yadav
Jyoti
J
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan
Sagami
Ikuko
I
Shimizu
Toru
T
D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D001665:Binding Sites; D002956:Citrulline / Q000031:analogs & derivatives* / Q000494:pharmacology; D003486:Cyanides / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007191:Indazoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D013056:Spectrophotometry, Ultraviolet; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
D003486:Cyanides; D004791:Enzyme Inhibitors; D007191:Indazoles; C086715:thiocitrulline; D002956:Citrulline; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D013890:Thiourea; C080122:7-nitroindazole; D019331:NG-Nitroarginine Methyl Ester