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 enzyme s that catalyze the formation of nitric oxide from L-Arg, General cyto chrome P-450 inhibitors and cytochrome P-450 isoform-selective substra tes and inhibitors were used to characterize the activity of recombina nt human inducible NOS (iNOS). Classical cytochrome P-450 ligands such as the mechanism-based inactivator 1-aminobenzotriazole did not inhib it iNOS. Of a panel of 30 human cytochrome P-450 isoform selective sub strates and inhibitors, only chlorzoxazone, a cytochrome P-450 2E1 (CY P2E1) substrate, showed any significant inhibition of iNOS activity. C hlorzoxazone was not a substrate for iNOS but was a potent competitive inhibitor with respect to L-Arg with K-i = 3.3 +/- 0.7 mu M. The bind ing of chlorzoxazone to iNOS and human and rat liver microsomal cytoch rome P-450 induced a high spin, type I spectra, which was reversed by imidazole, Although the binding of chlorzoxazone to iNOS and its inhib ition of iNOS activity suggest some similarity between iNOS and CYP2E1 activity, other CYP2E1 substrates and inhibitors including zoxazolami ne were not inhibitors of iNOS. Overall, iNOS activity is distinctly d ifferent from the major cytochrome P-450 enzymes in human liver micros omes.