Mechanisms of Mammalian Nitric Oxide Biosynthesis

Abstract

In mammals nitric oxide (NO) synthesis is catalyzed by the enzyme(s) nitric oxide synthase (NOS).¹ NOS oxidizes one of the two equivalent terminal guanidino nitrogens of the protogenic amino acid L-arginine (Arg) to yield NO and the co-product L-citrulline (Cit).² NOS constitutes a family of enzymes of which two distinct types, inducible NOS (iNOS) and constitutive NOS (cNOS), have been cloned and characterized. Both iNOS and cNOS are dimeric enzymes containing two identical subunits with molecular weights in the range of 130–150 k D. They have similar specific activities of about 0.8–1.3 µmole/min-mg of protein at 37°C with a turnover rate of about 2.5–3.2 molecules/sec for each subunit. Both forms of NOS are dependent upon the cofactors tetrahydrobiopterin (BH4), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and heme for enzymatic activity. Each subunit contains one molecule of each of the flavins, heme, and BH4. Although all four of these cofactors are widely utilized by various enzymes in nature to catalyze various oxidative and reductive reactions, NOS is the only known mammalian enzyme to contain all four groups. Given the importance of understanding the physiology and biochemistry of NO-mediated processes and translating that understanding to therapeutic benefit, we have studied NOS enzymology with the goal to determine the mechanism of NO biosynthesis.