Mechanistic Studies of the Inactivation of Inducible Nitric Oxide Synthase by N5-(1-Iminoethyl)- l -ornithine ( l -NIO)

Nitric oxide synthase (NOS) catalyzes the conversion of l -arginine to l -citrulline and nitric oxide. N 5 -(1-Iminoethyl)- l -ornithine ( l -NIO, 5 ) is a natural product known to inactivate NOS, but the mechanism of inactivation is unknown. Upon incubation of iNOS with l -NIO a type I binding difference spectrum is observed, indicating that binding at the substrate binding site occurs. l -NIO is shown to be a time-dependent, concentration-dependent, and NADPH-dependent irreversible inhibitor of iNOS with K I and k inact values of 13.7 ± 1.6 μM and 0.073 ± 0.003 min - 1, respectively. During inactivation the heme chromophore is partially lost (Figure 1); HPLC shows that the loss corresponds to about 50% of the heme. Inclusion of catalase during incubation does not prevent heme loss. N 5 -(1-Imino-2-[ 14 C]ethyl)- l -ornithine ( 11 ) inactivates iNOS, but upon dialysis or gel filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected after enzyme inactivation is N ω -hydroxy- l -NIO ( 12 ); no C ω -hydroxy- l -NIO ( 13 ) or N δ -acetyl- l -ornithine ( 14 ) is observed (Figure 2). The amount of 12 produced during the inactivation process is 7.7 ± 0.2 equiv per inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation that is not reversible upon dilution into the assay solution. Incubations that include an excess of l -arginine or with substitution of NADP + for NADPH result in no significant loss of enzyme activity. The K I and k inact values for 12 are 830 ± 160 μM and 0.0073 ± 0.0007 min - 1, respectively. The magnitude of these kinetic constants (compared with those of 5 ) suggest that 12 is not an intermediate of l -NIO inactivation of iNOS. Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with K m and k cat values of 800 ± 85 μM and 2.22 min - 1, respectively; the product is shown to be N δ -acetyl- l -ornithine ( 14 ) (Figure 3). The k cat and k inact values for 12 can be compared directly to give a partition ratio ( k cat / k inact ) for inactivation of 304; i.e., there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion that 12 is not involved in l -NIO inactivation of iNOS. C ω -Hydroxy- l -NIO ( 13 ) is not an inactivator of iNOS. These results suggest that l -NIO inactivation occurs after an oxidation step (NADPH is required for inactivation) but prior to a hydroxylation step ( 12 and 13 are not involved). Inactivation of iNOS by N 5 -(1-imino-2-[ 2 H 3 ]ethyl)- l -ornithine ( 15 ) exhibits a kinetic isotope effect on H k inact / D k inact of 1.35 ± 0.08 and on H ( k inact / K I )/ D ( k inact / K I ) of 1.51 ± 0.3, suggesting that the methyl C−H bond is cleaved in a partially rate-determining step prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of l -NIO-inactivated iNOS is produced (Figure 4). LC−electrospray mass spectrometry (Figure 5) demonstrates the m / z of the new metabolite to be 583, which is shown to correspond to biliverdin ( 23 ) (Figures 6 and 7). Two possible mechanisms for the formation of biliverdin during inactivation are proposed (Schemes 10 and 11). When 14 is incubated with iNOS, time-, concentration-, and NADPH-dependent loss of enzyme activity is observed ( K I and k inact values are 490 mM and 0.24 min - 1, respectively); iNOS inactivation by 14 can be prevented by inclusion of l -arginine, but not d -arginine, in the inactivation mixtures, suggesting that the inactivator acts at the arginine binding site. However, 14 is not produced from l -NIO (Figure 2) and, therefore, is not involved in l -NIO inactivation.