Mechanism of inducible nitric oxide synthase inactivation by aminoguanidine and L-N6-(1-iminoethyl)lysine.
The inducible nitric oxide synthase (iNOS) selective inhibitors aminoguanidine (AG) and N6-(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 fluorescence that accompanies the loss of NO-forming activity. The NIL-inactivated iNOS dimers that have lost their heme partially disassemble into monomers with no fluorometrically detectable heme. AG inactivation of the iNOS is not accompanied by heme destruction, as evidenced by retention of heme fluorescence and absorbance after complete loss of NO-forming activity. The AG-inactivated iNOS dimers do not disassemble into monomers as extensively as NIL-inactivated dimers. Incubation of the iNOS with 14C-labeled NIL results in no detectable protein-associated radioactivity 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 14C-labeled AG result in the incorporation of radioactivity into both iNOS protein and low molecular weight structures that migrate by SDS-PAGE similarly to free heme. These observations suggest that AG inactivation proceeds through multiple pathways of covalent modification of the iNOS protein and the heme residue at the active site, but which sustain the integrity of the heme porphyrin ring.
Bryk
R
R
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. bryk@umdnj.edu
Wolff
D J
DJ
D000818:Animals; D002247:Carbon Isotopes; D002851:Chromatography, High Pressure Liquid; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006146:Guanidines / Q000494:pharmacology*; D007700:Kinetics; D008239:Lysine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D013050:Spectrometry, Fluorescence
D002247:Carbon Isotopes; D004791:Enzyme Inhibitors; D006146:Guanidines; C090663:N(6)-(1-iminoethyl)lysine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D008239:Lysine; C004479:pimagedine