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. 1997 Jan 15;321(Pt 2):503–508. doi: 10.1042/bj3210503

Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide.

A J Kettle 1, C A Gedye 1, C C Winterbourn 1
PMCID: PMC1218097  PMID: 9020887

Abstract

Hypochlorous acid is the most powerful oxidant generated by neutrophils and is likely to contribute to the damage mediated by these inflammatory cells. The haem enzyme myeloperoxidase catalyses its production from hydrogen peroxide and chloride. 4-Aminobenzoic acid hydrazide (ABAH) is a potent inhibitor of hypochlorous acid production. In this investigation we show that, in the presence of hydrogen peroxide, ABAH irreversibly inactivates myeloperoxidase. ABAH was oxidized by myeloperoxidase, and kinetic analysis of the inactivation conformed to that for a mechanism-based inhibitor. Inactivation was exacerbated by concentrations of hydrogen peroxide greater than 50 microM and by the absence of oxygen. Hydrogen peroxide alone caused minimal inactivation. Reduced glutathione inhibited the oxidation of ABAH as well as the irreversible inhibition of myeloperoxidase. In the presence of oxygen, ABAH and hydrogen peroxide initially converted myeloperoxidase into compound III, which subsequently lost haem absorbance. In the absence of oxygen, the enzyme was converted into ferrous myeloperoxidase and its haem groups were rapidly destroyed. We propose that myeloperoxidase oxidizes ABAH to a radical that reduces the enzyme to its ferrous intermediate. Ferrous myeloperoxidase reacts either with oxygen to allow enzyme turnover, or with hydrogen peroxide to give irreversible inactivation.

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Selected References

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  1. Albrich J. M., McCarthy C. A., Hurst J. K. Biological reactivity of hypochlorous acid: implications for microbicidal mechanisms of leukocyte myeloperoxidase. Proc Natl Acad Sci U S A. 1981 Jan;78(1):210–214. doi: 10.1073/pnas.78.1.210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ator M. A., David S. K., Ortiz de Montellano P. R. Structure and catalytic mechanism of horseradish peroxidase. Regiospecific meso alkylation of the prosthetic heme group by alkylhydrazines. J Biol Chem. 1987 Nov 5;262(31):14954–14960. [PubMed] [Google Scholar]
  3. Ator M. A., Ortiz de Montellano P. R. Protein control of prosthetic heme reactivity. Reaction of substrates with the heme edge of horseradish peroxidase. J Biol Chem. 1987 Feb 5;262(4):1542–1551. [PubMed] [Google Scholar]
  4. BEERS R. F., Jr, SIZER I. W. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem. 1952 Mar;195(1):133–140. [PubMed] [Google Scholar]
  5. Bozeman P. M., Learn D. B., Thomas E. L. Assay of the human leukocyte enzymes myeloperoxidase and eosinophil peroxidase. J Immunol Methods. 1990 Jan 24;126(1):125–133. doi: 10.1016/0022-1759(90)90020-v. [DOI] [PubMed] [Google Scholar]
  6. Davies B., Edwards S. W. Inhibition of myeloperoxidase by salicylhydroxamic acid. Biochem J. 1989 Mar 15;258(3):801–806. doi: 10.1042/bj2580801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Foote C. S., Goyne T. E., Lehrer R. I. Assessment of chlorination by human neutrophils. Nature. 1983 Feb 24;301(5902):715–716. doi: 10.1038/301715a0. [DOI] [PubMed] [Google Scholar]
  8. Harrison J. E., Schultz J. Studies on the chlorinating activity of myeloperoxidase. J Biol Chem. 1976 Mar 10;251(5):1371–1374. [PubMed] [Google Scholar]
  9. Hoogland H., van Kuilenburg A., van Riel C., Muijsers A. O., Wever R. Spectral properties of myeloperoxidase compounds II and III. Biochim Biophys Acta. 1987 Nov 5;916(1):76–82. doi: 10.1016/0167-4838(87)90212-3. [DOI] [PubMed] [Google Scholar]
  10. Ikeda-Saito M., Shelley D. A., Lu L., Booth K. S., Caughey W. S., Kimura S. Salicylhydroxamic acid inhibits myeloperoxidase activity. J Biol Chem. 1991 Feb 25;266(6):3611–3616. [PubMed] [Google Scholar]
  11. Jenzer H., Kohler H., Broger C. The role of hydroxyl radicals in irreversible inactivation of lactoperoxidase by excess H2O2. A spin-trapping/ESR and absorption spectroscopy study. Arch Biochem Biophys. 1987 Nov 1;258(2):381–390. doi: 10.1016/0003-9861(87)90359-6. [DOI] [PubMed] [Google Scholar]
  12. Karuzina I. I., Archakov A. I. The oxidative inactivation of cytochrome P450 in monooxygenase reactions. Free Radic Biol Med. 1994 Jan;16(1):73–97. doi: 10.1016/0891-5849(94)90245-3. [DOI] [PubMed] [Google Scholar]
  13. Kettle A. J., Gedye C. A., Hampton M. B., Winterbourn C. C. Inhibition of myeloperoxidase by benzoic acid hydrazides. Biochem J. 1995 Jun 1;308(Pt 2):559–563. doi: 10.1042/bj3080559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kettle A. J., Gedye C. A., Winterbourn C. C. Superoxide is an antagonist of antiinflammatory drugs that inhibit hypochlorous acid production by myeloperoxidase. Biochem Pharmacol. 1993 May 25;45(10):2003–2010. doi: 10.1016/0006-2952(93)90010-t. [DOI] [PubMed] [Google Scholar]
  15. Kettle A. J., Robertson I. G., Palmer B. D., Anderson R. F., Patel K. B., Winterbourn C. C. Oxidative metabolism of amsacrine by the neutrophil enzyme myeloperoxidase. Biochem Pharmacol. 1992 Nov 3;44(9):1731–1738. doi: 10.1016/0006-2952(92)90066-r. [DOI] [PubMed] [Google Scholar]
  16. Kettle A. J., Winterbourn C. C. Assays for the chlorination activity of myeloperoxidase. Methods Enzymol. 1994;233:502–512. doi: 10.1016/s0076-6879(94)33056-5. [DOI] [PubMed] [Google Scholar]
  17. Kettle A. J., Winterbourn C. C. Mechanism of inhibition of myeloperoxidase by anti-inflammatory drugs. Biochem Pharmacol. 1991 May 15;41(10):1485–1492. doi: 10.1016/0006-2952(91)90565-m. [DOI] [PubMed] [Google Scholar]
  18. Kettle A. J., Winterbourn C. C. Oxidation of hydroquinone by myeloperoxidase. Mechanism of stimulation by benzoquinone. J Biol Chem. 1992 Apr 25;267(12):8319–8324. [PubMed] [Google Scholar]
  19. Kettle A. J., Winterbourn C. C. Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid. Biochem J. 1988 Jun 1;252(2):529–536. doi: 10.1042/bj2520529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee E., Miki Y., Katsura H., Kariya K. Mechanism of inactivation of myeloperoxidase by propylthiouracil. Biochem Pharmacol. 1990 May 1;39(9):1467–1471. doi: 10.1016/0006-2952(90)90428-n. [DOI] [PubMed] [Google Scholar]
  21. O'Brien P. J. Radical formation during the peroxidase catalyzed metabolism of carcinogens and xenobiotics: the reactivity of these radicals with GSH, DNA, and unsaturated lipid. Free Radic Biol Med. 1988;4(3):169–183. doi: 10.1016/0891-5849(88)90025-1. [DOI] [PubMed] [Google Scholar]
  22. Odajima T., Yamazaki I. Myeloneperoxidase of the leukocyte of normal blood. 3. The reaction of ferric myeloperoxidase with superoxide anion. Biochim Biophys Acta. 1972 Oct 12;284(2):355–359. doi: 10.1016/0005-2744(72)90130-1. [DOI] [PubMed] [Google Scholar]
  23. Odajima T., Yamazaki I. Myeloperoxidase of the leukocyte of normal blood. I. Reaction of myeloperoxidase with hydrogen peroxide. Biochim Biophys Acta. 1970 Apr 22;206(1):71–77. doi: 10.1016/0005-2744(70)90083-5. [DOI] [PubMed] [Google Scholar]
  24. Ortiz de Montellano P. R., David S. K., Ator M. A., Tew D. Mechanism-based inactivation of horseradish peroxidase by sodium azide. Formation of meso-azidoprotoporphyrin IX. Biochemistry. 1988 Jul 26;27(15):5470–5476. doi: 10.1021/bi00415a013. [DOI] [PubMed] [Google Scholar]
  25. Segal A. W. The molecular and cellular pathology of chronic granulomatous disease. Eur J Clin Invest. 1988 Oct;18(5):433–443. doi: 10.1111/j.1365-2362.1988.tb01037.x. [DOI] [PubMed] [Google Scholar]
  26. Shacter E., Lopez R. L., Pati S. Inhibition of the myeloperoxidase-H2O2-Cl- system of neutrophils by indomethacin and other non-steroidal anti-inflammatory drugs. 1991 Mar 15-Apr 1Biochem Pharmacol. 41(6-7):975–984. doi: 10.1016/0006-2952(91)90204-i. [DOI] [PubMed] [Google Scholar]
  27. Sinha B. K. Enzymatic activation of hydrazine derivatives. A spin-trapping study. J Biol Chem. 1983 Jan 25;258(2):796–801. [PubMed] [Google Scholar]
  28. Svensson B. E. Thiols as myeloperoxidase-oxidase substrates. Biochem J. 1988 Jul 15;253(2):441–449. doi: 10.1042/bj2530441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Waley S. G. Kinetics of suicide substrates. Practical procedures for determining parameters. Biochem J. 1985 May 1;227(3):843–849. doi: 10.1042/bj2270843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Winterbourn C. C. Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim Biophys Acta. 1985 Jun 18;840(2):204–210. doi: 10.1016/0304-4165(85)90120-5. [DOI] [PubMed] [Google Scholar]
  31. Winterbourn C. C., Garcia R. C., Segal A. W. Production of the superoxide adduct of myeloperoxidase (compound III) by stimulated human neutrophils and its reactivity with hydrogen peroxide and chloride. Biochem J. 1985 Jun 15;228(3):583–592. doi: 10.1042/bj2280583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Zyl J. M., Basson K., Uebel R. A., van der Walt B. J. Isoniazid-mediated irreversible inhibition of the myeloperoxidase antimicrobial system of the human neutrophil and the effect of thyronines. Biochem Pharmacol. 1989 Jul 15;38(14):2363–2373. doi: 10.1016/0006-2952(89)90477-2. [DOI] [PubMed] [Google Scholar]
  33. van der Walt B. J., van Zyl J. M., Kriegler A. Aromatic hydroxylation during the myeloperoxidase-oxidase oxidation of hydrazines. Biochem Pharmacol. 1994 Mar 15;47(6):1039–1046. doi: 10.1016/0006-2952(94)90415-4. [DOI] [PubMed] [Google Scholar]

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