N⁵-(1-imino-2-hydroxyethyl)-L-ornithine

• 分子结构分类: 有机化合物 - 有机酸及其衍生物 - 羧酸及其衍生物
• 英文名称: N⁵-(1-imino-2-hydroxyethyl)-L-ornithine
• 英文别名: (S)-2-Amino-5-(2-Hydroxyacetimidamido)pentanoic Acid；(2S)-2-amino-5-[(1-amino-2-hydroxyethylidene)amino]pentanoic acid
• 化学式: C₇H₁₅N₃O₃
• 分子量: 189.214
• InChiKey: TXBVSXZGQDGYCU-YFKPBYRVSA-N

计算性质

• 辛醇/水分配系数(LogP)：
  -4.3
• 重原子数：
  13
• 可旋转键数：
  6
• 环数：
  0.0
• sp3杂化的碳原子比例：
  0.71
• 拓扑面积：
  122
• 氢给体数：
  4
• 氢受体数：
  5

反应信息

• 作为产物：
  描述：

  N^α-Boc-N⁵-(1-imino-2-hydroxyethyl)-L-ornithine 在 盐酸 作用下， 以 乙酸乙酯 为溶剂， 反应 1.0h， 以92%的产率得到N⁵-(1-imino-2-hydroxyethyl)-L-ornithine
  参考文献：
  名称：

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

  摘要：

  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 mu M 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-[C-14]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 Nw-hydroxy-L-NIO (12); no C-omega-hydroxy-L-NIO (13) or N-delta-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 an 830 +/- 160 mu M 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 mu M and 2.22 min(-1), respectively; the product is shown to be N-delta-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-omega-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-[H-2(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 mit 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.

  DOI：

  10.1021/ja982318l

文献信息

• Mechanistic Studies of the Inactivation of Inducible Nitric Oxide Synthase by <i>N</i>⁵-(1-Iminoethyl)-<scp>l</scp>-ornithine (<scp>l</scp>-NIO)
  作者：Walter Fast、Dejan Nikolic、Richard B. Van Breemen、Richard B. Silverman

  DOI：10.1021/ja982318l

  日期：1999.2.1

  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 mu M 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-[C-14]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 Nw-hydroxy-L-NIO (12); no C-omega-hydroxy-L-NIO (13) or N-delta-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 an 830 +/- 160 mu M 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 mu M and 2.22 min(-1), respectively; the product is shown to be N-delta-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-omega-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-[H-2(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 mit 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.