Characterization of inducible nitric-oxide synthase by cytochrome P-450 substrates and inhibitors. Inhibition by chlorzoxazone.
Nitric-oxide synthases (NOS, EC 1.14.13.39) are heme-containing enzymes that catalyze the formation of nitric oxide from L-Arg. General cytochrome P-450 inhibitors and cytochrome P-450 isoform-selective substrates and inhibitors were used to characterize the activity of recombinant human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhibit iNOS. Of a panel of 30 human cytochrome P-450 isoform-selective substrates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CYP2E1) substrate, showed any significant inhibition of iNOS activity. Chlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with Ki = 3.3+/-0.7 microM. The binding of chlorzoxazone to iNOS and human and rat liver microsomal cytochrome P-450 induced a high spin, type I spectra, which was reversed by imidazole. Although the binding of chlorzoxazone to iNOS and its inhibition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolamine were not inhibitors of iNOS. Overall, iNOS activity is distinctly different from the major cytochrome P-450 enzymes in human liver microsomes.
Grant
S K
SK
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
Green
B G
BG
Wang
R
R
Pacholok
S G
SG
Kozarich
J W
JW
D000818:Animals; D002753:Chlorzoxazone / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004790:Enzyme Induction; D006427:Hemin / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007700:Kinetics; D009125:Muscle Relaxants, Central / Q000494:pharmacology*; D009249:NADP / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D051381:Rats
D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D009125:Muscle Relaxants, Central; D009249:NADP; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D019392:Cytochrome P-450 CYP2E1; D019001:Nitric Oxide Synthase; D002753:Chlorzoxazone