Deuterium isotope effects and product studies for the oxidation of N(ω)-allyl-L-arginine and N(ω)-allyl-N(ω)-hydroxy-L-arginine by neuronal nitric oxide synthase

The nitric oxide synthases (NOS), which require heme, tetrahydrobiopterin, FMN, FAD, and NADPH, catalyze the O₂-dependent conversion of L-arginine to L-citrulline and nitric oxide. N(ω)-Allyl-L-arginine, a mechanism-based inactivator of neuronal NOS, also is a substrate, producing L-arginine, acrolein, and H₂O (Zhang, H. Q.; Dixon, R. P.; Marletta, M. A.; Nikolic, D.; Van Breemen, R.; Silverman, R. B. J. Am. Chem. Soc. 1997, 119, 10888). Two possible mechanisms for this turnover are proposed, one initiated by allyl C-H bond cleavage and the other by guanidino N-H cleavage, and these mechanisms are investigated with the use of N(ω)-allyl-L-arginine (1), N(ω)-[1,1-²H₂]allyl-L-arginine (7), N(ω)-allyl-N(ω)-hydroxy-L-arginine (2) and N(ω)-[1,1-²H₂]allyl-N(ω)-hydroxy-L-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, k(cat) and k(cat)/K(m), are observed in case of 1 and 7 during turnover, but not with 2 and 8. No kinetic isotope effects are observed for either compound in their role as inactivators. These results support a mechanism involving initial C-H bond cleavage of N(ω)-allyl-L-arginine followed by hydroxylation and breakdown to products. Copyright (C) 2000.