Chiral recognition at the heme active site of nitric oxide synthase is markedly enhanced by L-arginine and 5,6,7,8-tetrahydrobiopterin.
The effects of substrate, L-Arg and cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the R/S ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 microM H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the Kd values for ligand I was decreased down to 5.9. In the presence of both 1 mM L-Arg and 0.1 mM H4B, the S/R ratios for ligands I and II and the R/S ratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.
Institute for Chemical Reaction Science, Tohoku University, Sendai, Japan.
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Nakano
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Sagami
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Daff
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Shimizu
D000693:Anaerobiosis; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001120:Arginine / Q000494:pharmacology*; D001665:Binding Sites; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D003001:Cloning, Molecular; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013053:Spectrophotometry; D013237:Stereoisomerism
D000975:Antioxidants; D002147:Calmodulin; D011994:Recombinant Proteins; D001708:Biopterins; D006418:Heme; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat; C003402:sapropterin