Deuterium isotope effects and product studies for the oxidation of N(omega)-allyl-L-arginine and N(omega)-allyl-N(omega)-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(omega)-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(omega)-allyl-L-arginine (1), N(omega)-[1,1-(2)H2]allyl-L-arginine (7), N(omega)-allyl-N(omega)-hydroxy-L-arginine (2) and N(omega)-[1,1-(2)H2]allyl-N(omega)-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 N(omega)-allyl-L-arginine followed by hydroxylation and breakdown to products.
Hah
J M
JM
Department of Chemistry, Northwestern University, Evanston, IL 60208-3113, USA.
Roman
L J
LJ
Silverman
R B
RB
D000171:Acrolein / Q000378:metabolism; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry*; D006900:Hydroxylation; D007700:Kinetics; D015394:Molecular Structure; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins; D021241:Spectrometry, Mass, Electrospray Ionization
D011994:Recombinant Proteins; C073753:omega-N-allylarginine; D000171:Acrolein; D001120:Arginine; D003903:Deuterium; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat