MECHANISM OF INDUCIBLE NITRIC-OXIDE SYNTHASE INACTIVATION BY AMINOGUANIDINE AND L-N-6-(1-IMINOETHYL)LYSINE

The inducible nitric oxide synthase (iNOS) selective inhibitors aminog uanidine (AG) and N-6-(1-iminoethyl)-L-lysine (NIL), under conditions that support catalytic turnover, inactivate the enzyme by altering in different ways the functionality of the active site. NIL inactivation of the iNOS primarily targets the heme residue at the active site, as evidenced by a time-and concentration-dependent loss of heme fluoresce nce that accompanies the loss of NO-forming activity. The NIL-inactiva ted iNOS dimers that have lost their heme partially disassemble into m onomers with no fluorometrically detectable heme. AG inactivation of t he iNOS is not accompanied by heme destruction, as evidenced by retent ion of heme fluorescence and absorbance after complete loss of NO-form ing activity. The AG-inactivated iNOS dimers do not disassemble into m onomers as extensively as NIL-inactivated dimers, Incubation of the iN OS with C-14-labeled NIL results in no detectable protein-associated r adioactivity in the NIL-inactivated iNOS, suggesting that the primary mechanism of the iNOS inactivation by NIL is heme alteration and loss. In contrast, incubations of iNOS with C-14-labeled AG result in the i ncorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. Th ese observations suggest that AG inactivation proceeds through multipl e pathways of covalent modification of the iNOS protein and the heme r esidue at the active site, but which sustain the integrity of the heme porphyrin ring.