Mechanistic probes of N-hydroxylation of L-arginine by the inducible nitric oxide synthase from murine macrophages.
NG-Hydroxy-L-arginine, [15N]-NG-hydroxy-L-arginine, and NG-hydroxy-NG- methyl-L-arginine were used as mechanistic probes of the initial step in the reaction catalyzed by nitric oxide synthase isolated from murine macrophages. NG-Hydroxy-L-arginine was found to be a substrate for nitric oxide synthase with a Km equal to 28.0 microM, 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 NG-hydroxy-L-arginine loss to L-citrulline and nitric oxide (measured as nitrite and nitrate) formation was found to be 1:1:1. NG-Hydroxy-L-arginine was also observed in small amounts from L-arginine during the enzyme reaction. Studies with [15N]-NG-hydroxy-L-arginine indicated that the nitrogen in nitric oxide is derived from the oxime nitrogen of [15N]-NG-hydroxy-L- arginine. NG-Hydroxy-NG-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 microM. As an irreversible inhibitor, NG-hydroxy-NG-methyl-L-arginine gave kinact equal to 0.16 min-1 and KI equal to 26.5 microM. This inhibition was found to be both time- and concentration-dependent as well as showing substrate protection against inactivation. Gel filtration of an NG-hydroxy-NG-methyl-L-arginine-inactivated nitric oxide synthase failed to recover substantial amounts of enzyme activity.
Pufahl
R A
RA
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Nanjappan
P G
PG
Woodard
R W
RW
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism* / Q000494:pharmacology; D006900:Hydroxylation; D007202:Indicators and Reagents; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D019001:Nitric Oxide Synthase; D013379:Substrate Specificity
D007202:Indicators and Reagents; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases