N-5-(1-IMINO-5-BUTENYL)-L-ORNITHINE - A NEURONAL ISOFORM SELECTIVE MECHANISM-BASED INACTIVATOR OF NITRIC-OXIDE SYNTHASE

Nitric oxide synthase (NOS) catalyzes the NADPH-and O-2-dependent conv ersion of L-arginine to nitric oxide (NO) and citrulline; three isofor ms, the neuronal (nNOS), endothelial, and inducible, have been identif ied. Because overproduction of NO is known to contribute to several pa thophysiological conditions, NOS inhibitors are of interest as potenti al therapeutic agents, Inhibitors that are potent, mechanism-based, an d relatively selective for the NOS isoform causing pathology are of pa rticular interest, In the present studies we report that vinyl-L-NIO ( N-5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNO S in competition with L-arginine (K-i = 100 nM); binding is accompanie d by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. S uch binding is fully reversible, However, in the presence of NADPH and O-2, L-VNIO irreversibly inactivates nNOS (K-inact = 0.078 min(-1); K -I = 90 nM); inactivation is Ca2+/calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish th at the nNOS heme cofactor is lost or modified by L-VNIO-mediated mecha nism-based inactivation of the enzyme, The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fo ld higher concentrations to attain similar to 75% of the rate of inact ivation seen with nNOS, Among the NOS inactivating L-arginine derivati ves, L-VNIO is the most potent and nNOS-selective reported to date.