Characterization of the inactivation of nitric oxide synthase by NG-methyl-L-arginine: evidence for heme loss.
The nitric oxide synthases (NOS) are a unique family of P450-type hemoproteins that catalyze the formation of .NO and citrulline from L-arginine, oxygen, and NADPH. NG-Methyl-L-arginine (L-NMA) has been shown to function as a slow, partially uncoupled alternate substrate and mechanism-based inhibitor of the inducible NOS [Olken, N. M., & Marletta, M. A. (1993) Biochemistry 32, 9677-9685]. In this report, the inactivation of NOS by L-NMA has been investigated in detail. Inactivation fails to occur under an argon atmosphere, establishing turnover dependence. The partition ratio, defined as the number of molecules of citrulline formed per NOS monomer inactivated, is 108 +/- 3. By utilizing NG-methyl-L-[2,3-3H2]arginine and NG-[14C]methyl-L-arginine, the stoichiometry of radiolabeling is 0.11 +/- 0.01 equiv of tritium and 0.41 +/- 0.10 equiv of carbon-14 per inactivated NOS monomer. Dialysis under native conditions does not change this stoichiometry. However, dialysis of NOS following denaturation decreases the stoichiometry of radiolabeling to 0.08 +/- 0.04 equiv of tritium and 0.12 +/- 0.04 equiv of carbon-14 per inactivated NOS monomer. Absolute and CO-reduced difference spectroscopy indicates that inactivation of L-NMA is accompanied by a substantial loss of the heme chromophore, which is not prevented by catalase. HPLC analysis of NOS heme following inactivation with L-NMA indicates substantial loss of heme. These findings suggest that multiple mechanisms may contribute to the loss of NOS activity by L-NMA, including heme loss and possibly protein and cofactor modification.
Olken
N M
NM
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065.
Osawa
Y
Y
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D002250:Carbon Radioisotopes / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D002956:Citrulline / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism*; D006420:Hemeproteins / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007553:Isotope Labeling; D019001:Nitric Oxide Synthase; D012995:Solubility; D013053:Spectrophotometry; D014316:Tritium / Q000737:chemistry; D019323:omega-N-Methylarginine
D002250:Carbon Radioisotopes; D006420:Hemeproteins; D014316:Tritium; D019323:omega-N-Methylarginine; D002956:Citrulline; D006418:Heme; C048576:omega-N-hydroxymethylarginine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D006861:Hydrogen Peroxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases