Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Nov 1;335(Pt 3):701–709. doi: 10.1042/bj3350701

Inactivation of papain by antithrombin due to autolytic digestion: a model of serpin inactivation of cysteine proteinases.

I Björk 1, K Nordling 1, E Raub-Segall 1, U Hellman 1, S T Olson 1
PMCID: PMC1219835  PMID: 9794814

Abstract

Cross-class inhibition of cysteine proteinases by serpins differs from serpin inhibition of serine proteinases primarily in that no stable serpin-cysteine proteinase complex can be demonstrated. This difference in reaction mechanism was elucidated by studies of the inactivation of the cysteine proteinases, papain and cathepsin L, by the serpin antithrombin. The two proteinases were inactivated with second-order rate constants of (1.6+/-0.1)x10(3) and (8.6+/-0. 4)x10(2) M-1.s-1 respectively. An antithrombin to papain inactivation stoichiometry of approximately 3 indicated extensive cleavage of the inhibitor concurrent with enzyme inactivation, a behaviour verified by SDS/PAGE. N-terminal sequence analyses showed cleavage predominantly at the P2-P1 bond, but also at the P2'-P3' bond of antithrombin. The papain band in SDS/PAGE progressively disappeared on reaction of the enzyme with increasing amounts of antithrombin, but no band representing a stable antithrombin-papain complex appeared. SDS/PAGE with 125I-labelled papain showed that the disappearance of papain was caused by cleavage of the enzyme into small fragments. These results suggest a mechanism in which papain attacks a peptide bond in the reactive-bond loop of antithrombin adjacent to that involved in serine proteinase inhibition. The reaction proceeds, similarly to that between serpins and serine proteinases, to form an inactive acyl-intermediate complex, although with the substrate pathway dominating in the papain reaction. In this complex, papain is highly susceptible to proteolysis and is degraded by still active papain, which greatly decreases the lifetime of the complex and results in liberation of fragmented, inactive enzyme. This model may have relevance also for the inactivation of physiologically or pathologically important cysteine proteinases by serpins.

Full Text

The Full Text of this article is available as a PDF (497.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Björk I., Nordling K., Olson S. T. Immunologic evidence for insertion of the reactive-bond loop of antithrombin into the A beta-sheet of the inhibitor during trapping of target proteinases. Biochemistry. 1993 Jul 6;32(26):6501–6505. doi: 10.1021/bi00077a002. [DOI] [PubMed] [Google Scholar]
  2. Bock S. C., Wion K. L., Vehar G. A., Lawn R. M. Cloning and expression of the cDNA for human antithrombin III. Nucleic Acids Res. 1982 Dec 20;10(24):8113–8125. doi: 10.1093/nar/10.24.8113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brubacher L. J., Bender M. L. The preparation and properties of trans-cinnamoyl-papain. J Am Chem Soc. 1966 Dec 20;88(24):5871–5880. doi: 10.1021/ja00976a032. [DOI] [PubMed] [Google Scholar]
  4. Bryan J. K. Molecular weights of protein multimers from polyacrylamide gel electrophoresis. Anal Biochem. 1977 Apr;78(2):513–519. doi: 10.1016/0003-2697(77)90111-7. [DOI] [PubMed] [Google Scholar]
  5. Carrell R. W., Stein P. E., Fermi G., Wardell M. R. Biological implications of a 3 A structure of dimeric antithrombin. Structure. 1994 Apr 15;2(4):257–270. doi: 10.1016/s0969-2126(00)00028-9. [DOI] [PubMed] [Google Scholar]
  6. Chang W. S., Wardell M. R., Lomas D. A., Carrell R. W. Probing serpin reactive-loop conformations by proteolytic cleavage. Biochem J. 1996 Mar 1;314(Pt 2):647–653. doi: 10.1042/bj3140647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choay J., Petitou M., Lormeau J. C., Sinaÿ P., Casu B., Gatti G. Structure-activity relationship in heparin: a synthetic pentasaccharide with high affinity for antithrombin III and eliciting high anti-factor Xa activity. Biochem Biophys Res Commun. 1983 Oct 31;116(2):492–499. doi: 10.1016/0006-291x(83)90550-8. [DOI] [PubMed] [Google Scholar]
  8. Cooperman B. S., Stavridi E., Nickbarg E., Rescorla E., Schechter N. M., Rubin H. Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex. J Biol Chem. 1993 Nov 5;268(31):23616–23625. [PubMed] [Google Scholar]
  9. Curtis M. A., Slaney J. M., Carman R. J., Pemberton P. A. Interaction of a trypsin-like enzyme of Porphyromonas gingivalis W83 with antithrombin III. FEMS Microbiol Lett. 1993 Apr 1;108(2):169–174. doi: 10.1111/j.1574-6968.1993.tb06094.x. [DOI] [PubMed] [Google Scholar]
  10. Dalet-Fumeron V., Guinec N., Pagano M. High-performance liquid chromatographic method for the simultaneous purification of cathepsins B, H and L from human liver. J Chromatogr. 1991 Jul 17;568(1):55–68. doi: 10.1016/0378-4347(91)80340-i. [DOI] [PubMed] [Google Scholar]
  11. Fish W. W., Björk I. Release of a two-chain form of antithrombin from the antithrombin-thrombin complex. Eur J Biochem. 1979 Nov 1;101(1):31–38. doi: 10.1111/j.1432-1033.1979.tb04212.x. [DOI] [PubMed] [Google Scholar]
  12. Fish W. W., Orre K., Björk I. Routes of thrombin action in the production of proteolytically modified, secondary forms of antithrombin-thrombin complex. Eur J Biochem. 1979 Nov 1;101(1):39–44. doi: 10.1111/j.1432-1033.1979.tb04213.x. [DOI] [PubMed] [Google Scholar]
  13. Hood D. B., Huntington J. A., Gettins P. G. Alpha 1-proteinase inhibitor variant T345R. Influence of P14 residue on substrate and inhibitory pathways. Biochemistry. 1994 Jul 19;33(28):8538–8547. doi: 10.1021/bi00194a020. [DOI] [PubMed] [Google Scholar]
  14. Hook V. Y., Purviance R. T., Azaryan A. V., Hubbard G., Krieger T. J. Purification and characterization of alpha 1-antichymotrypsin-like protease inhibitor that regulates prohormone thiol protease involved in enkephalin precursor processing. J Biol Chem. 1993 Sep 25;268(27):20570–20577. [PubMed] [Google Scholar]
  15. Kadowaki T., Yoneda M., Okamoto K., Maeda K., Yamamoto K. Purification and characterization of a novel arginine-specific cysteine proteinase (argingipain) involved in the pathogenesis of periodontal disease from the culture supernatant of Porphyromonas gingivalis. J Biol Chem. 1994 Aug 19;269(33):21371–21378. [PubMed] [Google Scholar]
  16. Kaslik G., Patthy A., Bálint M., Gráf L. Trypsin complexed with alpha 1-proteinase inhibitor has an increased structural flexibility. FEBS Lett. 1995 Aug 21;370(3):179–183. doi: 10.1016/0014-5793(95)00816-r. [DOI] [PubMed] [Google Scholar]
  17. Komiyama T., Ray C. A., Pickup D. J., Howard A. D., Thornberry N. A., Peterson E. P., Salvesen G. Inhibition of interleukin-1 beta converting enzyme by the cowpox virus serpin CrmA. An example of cross-class inhibition. J Biol Chem. 1994 Jul 29;269(30):19331–19337. [PubMed] [Google Scholar]
  18. Lawrence D. A., Ginsburg D., Day D. E., Berkenpas M. B., Verhamme I. M., Kvassman J. O., Shore J. D. Serpin-protease complexes are trapped as stable acyl-enzyme intermediates. J Biol Chem. 1995 Oct 27;270(43):25309–25312. doi: 10.1074/jbc.270.43.25309. [DOI] [PubMed] [Google Scholar]
  19. Olson S. T., Björk I. Regulation of thrombin activity by antithrombin and heparin. Semin Thromb Hemost. 1994;20(4):373–409. doi: 10.1055/s-2007-1001928. [DOI] [PubMed] [Google Scholar]
  20. Olson S. T., Björk I., Shore J. D. Kinetic characterization of heparin-catalyzed and uncatalyzed inhibition of blood coagulation proteinases by antithrombin. Methods Enzymol. 1993;222:525–559. doi: 10.1016/0076-6879(93)22033-c. [DOI] [PubMed] [Google Scholar]
  21. Olson S. T., Bock P. E., Kvassman J., Shore J. D., Lawrence D. A., Ginsburg D., Björk I. Role of the catalytic serine in the interactions of serine proteinases with protein inhibitors of the serpin family. Contribution of a covalent interaction to the binding energy of serpin-proteinase complexes. J Biol Chem. 1995 Dec 15;270(50):30007–30017. doi: 10.1074/jbc.270.50.30007. [DOI] [PubMed] [Google Scholar]
  22. Olson S. T. Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin. J Biol Chem. 1985 Aug 25;260(18):10153–10160. [PubMed] [Google Scholar]
  23. Olson S. T., Sheffer R., Francis A. M. High molecular weight kininogen potentiates the heparin-accelerated inhibition of plasma kallikrein by antithrombin: role for antithrombin in the regulation of kallikrein. Biochemistry. 1993 Nov 16;32(45):12136–12147. doi: 10.1021/bi00096a026. [DOI] [PubMed] [Google Scholar]
  24. Pike R. N., Coetzer T. H., Dennison C. Proteolytically active complexes of cathepsin L and a cysteine proteinase inhibitor; purification and demonstration of their formation in vitro. Arch Biochem Biophys. 1992 May 1;294(2):623–629. doi: 10.1016/0003-9861(92)90734-e. [DOI] [PubMed] [Google Scholar]
  25. Schick C., Pemberton P. A., Shi G. P., Kamachi Y., Cataltepe S., Bartuski A. J., Gornstein E. R., Brömme D., Chapman H. A., Silverman G. A. Cross-class inhibition of the cysteine proteinases cathepsins K, L, and S by the serpin squamous cell carcinoma antigen 1: a kinetic analysis. Biochemistry. 1998 Apr 14;37(15):5258–5266. doi: 10.1021/bi972521d. [DOI] [PubMed] [Google Scholar]
  26. Schreuder H. A., de Boer B., Dijkema R., Mulders J., Theunissen H. J., Grootenhuis P. D., Hol W. G. The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions. Nat Struct Biol. 1994 Jan;1(1):48–54. doi: 10.1038/nsb0194-48. [DOI] [PubMed] [Google Scholar]
  27. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  28. Shore J. D., Day D. E., Francis-Chmura A. M., Verhamme I., Kvassman J., Lawrence D. A., Ginsburg D. A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem. 1995 Mar 10;270(10):5395–5398. doi: 10.1074/jbc.270.10.5395. [DOI] [PubMed] [Google Scholar]
  29. Skriver K., Wikoff W. R., Patston P. A., Tausk F., Schapira M., Kaplan A. P., Bock S. C. Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status. J Biol Chem. 1991 May 15;266(14):9216–9221. [PubMed] [Google Scholar]
  30. Stavridi E. S., O'Malley K., Lukacs C. M., Moore W. T., Lambris J. D., Christianson D. W., Rubin H., Cooperman B. S. Structural change in alpha-chymotrypsin induced by complexation with alpha 1-antichymotrypsin as seen by enhanced sensitivity to proteolysis. Biochemistry. 1996 Aug 20;35(33):10608–10615. doi: 10.1021/bi9605806. [DOI] [PubMed] [Google Scholar]
  31. Takeda A., Yamamoto T., Nakamura Y., Takahashi T., Hibino T. Squamous cell carcinoma antigen is a potent inhibitor of cysteine proteinase cathepsin L. FEBS Lett. 1995 Feb 6;359(1):78–80. doi: 10.1016/0014-5793(94)01456-b. [DOI] [PubMed] [Google Scholar]
  32. Tchoupe J. R., Moreau T., Gauthier F., Bieth J. G. Photometric or fluorometric assay of cathepsin B, L and H and papain using substrates with an aminotrifluoromethylcoumarin leaving group. Biochim Biophys Acta. 1991 Jan 8;1076(1):149–151. doi: 10.1016/0167-4838(91)90232-o. [DOI] [PubMed] [Google Scholar]
  33. Valeri A. M., Wilson S. M., Feinman R. D. Reaction of antithrombin with proteases. Evidence for a specific reaction with papain. Biochim Biophys Acta. 1980 Aug 7;614(2):526–533. doi: 10.1016/0005-2744(80)90241-7. [DOI] [PubMed] [Google Scholar]
  34. Zhou Q., Snipas S., Orth K., Muzio M., Dixit V. M., Salvesen G. S. Target protease specificity of the viral serpin CrmA. Analysis of five caspases. J Biol Chem. 1997 Mar 21;272(12):7797–7800. doi: 10.1074/jbc.272.12.7797. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES