MECHANISTIC PROBES OF N-HYDROXYLATION OF L-ARGININE BY THE INDUCIBLE NITRIC-OXIDE SYNTHASE FROM MURINE MACROPHAGES

N(G)-Hydroxy-L-arginine, [N-15]-N(G)-hydroxy-L-arginine, and N(G)-hydroxy-N(G)-methyl-L-arginine were used as mechanistic probes of the initial step in the reaction catalyzed by nitric oxide synthase isolated from murine macrophages. N(G)-Hydroxy-L-arginine was found to be a substrate for nitric oxide synthase with a K(m) equal to 28.0)mu-M, yielding nitric oxide and L-Citrulline. NADPH was required for the reaction and (6R)-tetrahydro-L-biopterin enhanced the initial rate of nitric oxide formation. The stoichiometry of N(G)-hydroxy-L-arginine loss to L-Citrulline and nitric oxide (measured as nitrite and nitrate) formation was found to be 1:1:1. N(G)-Hydroxy-L-arginine was also observed in small amounts from L-arginine during the enzyme reaction. Studies with [N-15]-N(G)-hydroxy-L-arginine indicated that the nitrogen in nitric oxide is derived from the oxime nitrogen of [N-15]-N(G)-hydroxy-L-arginine. N(G)-Hydroxy-N(G)methyl-L-arginine was found to be both a reversible and an irreversible inhibitor of nitric oxide synthase, displaying reversible competitive inhibition with K(i) equal to 33.5-mu-M. As an irreversible inhibitor, N(G)-hydroxy-N(G)-methyl-L-arginine gave k(inact) equal to 0.16 min-1 and K(I) equal to 26.5-mu-M. This inhibition was found to be both time- and concentration-dependent as well as showing substrate protection against inactivation. Gel filtration of an N(G)-hydroxy-N(G)-methyl-L-arginine-inactivated nitric oxide synthase failed to recover substantial amounts of enzyme activity.