Deuterium isotope effects and product studies for the oxidation of Nω-allyl-l-arginine and Nω-allyl-Nω-hydroxy-l-arginine by neuronal nitric oxide synthase

Abstract

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. Nω-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 Nω-allyl-l-arginine (1), Nω-[1,1-2H2]allyl-l-arginine (7), Nω-allyl-Nω-hydroxy-l-arginine (2) and Nω-[1,1-2H2]allyl-Nω-hydroxy-l-arginine (8) as substrates. Significant isotope effects on the two kinetic parameters, kcat and kcat/Km, 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 Nω-allyl-l-arginine followed by hydroxylation and breakdown to products.