Mechanistic probes of N-hydroxylation of L-arginine by the inducible nitric oxide synthase from murine macrophages

Biochemistry. 1992 Jul 28;31(29):6822-8. doi: 10.1021/bi00144a024.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Oxidoreductases / antagonists & inhibitors
  • Amino Acid Oxidoreductases / isolation & purification
  • Amino Acid Oxidoreductases / metabolism*
  • Animals
  • Arginine / analogs & derivatives*
  • Arginine / chemical synthesis
  • Arginine / metabolism*
  • Arginine / pharmacology
  • Hydroxylation
  • Indicators and Reagents
  • Kinetics
  • Macrophages / enzymology*
  • Magnetic Resonance Spectroscopy
  • Mice
  • Nitric Oxide Synthase
  • Substrate Specificity


  • Indicators and Reagents
  • Arginine
  • Nitric Oxide Synthase
  • Amino Acid Oxidoreductases