Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine.
.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.
Olken
N M
NM
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Rusche
K M
KM
Richards
M K
MK
Marletta
M A
MA
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001667:Binding, Competitive; D001853:Bone Marrow; D007700:Kinetics; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000201:enzymology*; D008706:Methemoglobin / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000378:metabolism; D013379:Substrate Specificity; D014508:Urea / Q000378:metabolism*; D019323:omega-N-Methylarginine
D010108:Oxyhemoglobins; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D014508:Urea; D008706:Methemoglobin; D001120:Arginine