Mechanistic studies of the inactivation of inducible nitric oxide synthase by N5-(1-iminoethyl)-L-ornithine (L-NIO)

Walter Fast, Dejan Nikolic, Richard B. Van Breemen, Richard B Silverman*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L- citrulline and nitric oxide. N5-(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 heine. Inclusion of catalase during incubation does not prevent heine loss. N5-(1-Imino-2-[14C]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 N5-(1-imino-2-[2H3]-ethyl)-L-ornithine (15) exhibits 3 kinetic isotope effect (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.

Original languageEnglish (US)
Pages (from-to)903-916
Number of pages14
JournalJournal of the American Chemical Society
Volume121
Issue number5
DOIs
StatePublished - Feb 10 1999

ASJC Scopus subject areas

  • Chemistry(all)

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