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 (Iigand 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 (K-d) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the RIS ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 mu M H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the K-d 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. (C) 1998 Academic Press.
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 30 Jul 1998|
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