Abstract
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.
Highlights
Nitric-oxide synthases (NOS, EC 1.14.13.39) are hemecontaining enzymes that catalyze the formation of nitric oxide from L-Arg
We have examined both general cytochrome P-450 inhibitors and a series of cytochrome P-450 isoform selective substrates and inhibitors against inducible NOS (iNOS) in order to characterize its activity compared with human cytochrome P-450s
Our results further demonstrate that iNOS activity is distinct from human cytochrome P-450 enzymes as evaluated by a panel of general and isoform-selective cytochrome P-450 substrates and inhibitors
Summary
Nitric-oxide synthases (NOS, EC 1.14.13.39) are hemecontaining 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). For iNOS, calmodulin is tightly associated with the enzyme and represents a distinguishing feature for the inducible versus the constitutively expressed mammalian NOS isoforms [10, 11] The interaction between these two catalytic domains means that NOS isoforms have within a single protein the complete catalytic machinery of the two-protein microsomal electron transfer system, reminiscent of the bacterial fatty acid monooxygenase cyctochrome P-450BM-3 [12, 13].
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