Chiral Recognition at the Heme Active Site of Nitric Oxide Synthase Is Markedly Enhanced byl-Arginine and 5,6,7,8-Tetrahydrobiopterin

Abstract

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 theS-andR-enantiomers of ligand I (S/R) was 30, while theS/Rratio for ligand II and theR/Sratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 μM H4B. However, in the presence of 1 mMl-Arg, theS/Rratio of theKdvalues for ligand I was decreased down to 5.9. In the presence of both 1 mMl-Arg and 0.1 mM H4B, theS/Rratios for ligands I and II and theR/Sratio 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.