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 discrimin ation by rat neuronal nitric oxide synthase (nNOS) for binding the ena ntiomers of 1-(1-naphthyl)ethylamine (Iigand I), 1-cyclohexylethylamin e (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied unde r anaerobic conditions by optical absorption spectroscopy. The ratio o f 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 RI S ratio for ligand III were 1.8 and < 0.14, respectively, in the prese nce 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 ligan ds 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 observ ations strongly suggest that strict chiral recognition at the active s ite of nNOS during catalysis is exhibited only in the presence of the active effector. (C) 1998 Academic Press.