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. 1996 Jun 15;316(Pt 3):777–786. doi: 10.1042/bj3160777

Demonstration that 1-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) is one of the most effective low Mr inhibitors of trypsin-catalysed hydrolysis. Characterization by kinetic analysis and by energy minimization and molecular dynamics simulation of the E-64-beta-trypsin complex.

S K Sreedharan 1, C Verma 1, L S Caves 1, S M Brocklehurst 1, S E Gharbia 1, H N Shah 1, K Brocklehurst 1
PMCID: PMC1217418  PMID: 8670152

Abstract

1-trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) was shown to inhibit beta-trypsin by a reversible competitive mechanism; this contrasts with the widely held view that E-64 is a class-specific inhibitor of the cysteine proteinases and reports in the literature that it does not inhibit a number of other enzymes including, notably, trypsin. The K1, value (3 x 10(-5) M) determined by kinetic analysis of the hydrolysis of N alpha-benzoyl-L-arginine 4-nitroanilide in Tris/HCl buffer, pH 7.4, at 25 degrees C, I = 0.1, catalysed by beta-trypsin is comparable with those for the inhibition of trypsin by benzamidine and 4-aminobenzamidine, which are widely regarded as the most effective low Mr inhibitors of this enzyme. Computer modelling of the beta-trypsin-E64 adsorptive complex, by energy minimization, molecular dynamics simulation and Poisson-Boltzmann electrostatic-potential calculations, was used to define the probable binding mode of E-64; the ligand lies parallel to the active-centre cleft, anchored principally by the dominant electrostatic interaction of the guanidinium cation at one end of the E-64 molecule with the carboxylate anion of Asp-171 (beta-trypsin numbering from Ile-1) in the S1-subsite, and by the interaction of the carboxylate substituent on C-2 of the epoxide ring at the other end of the molecule with Lys-43; the epoxide ring of E-64 is remote from the catalytic site serine hydroxy group. The possibility that E-64 might bind to the cysteine proteinases clostripain (from Clostridium histolyticum) and alpha-gingivain (one of the extracellular enzymes from phyromonas gingivalis) in a manner analogous to that deduced for the beta-trypsin-E-64 complex is discussed.

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Selected References

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  1. Barrett A. J., Kembhavi A. A., Brown M. A., Kirschke H., Knight C. G., Tamai M., Hanada K. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982 Jan 1;201(1):189–198. doi: 10.1042/bj2010189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartunik H. D., Summers L. J., Bartsch H. H. Crystal structure of bovine beta-trypsin at 1.5 A resolution in a crystal form with low molecular packing density. Active site geometry, ion pairs and solvent structure. J Mol Biol. 1989 Dec 20;210(4):813–828. doi: 10.1016/0022-2836(89)90110-1. [DOI] [PubMed] [Google Scholar]
  3. Berger A., Schechter I. Mapping the active site of papain with the aid of peptide substrates and inhibitors. Philos Trans R Soc Lond B Biol Sci. 1970 Feb 12;257(813):249–264. doi: 10.1098/rstb.1970.0024. [DOI] [PubMed] [Google Scholar]
  4. Bruccoleri R. E., Karplus M. Conformational sampling using high-temperature molecular dynamics. Biopolymers. 1990 Dec;29(14):1847–1862. doi: 10.1002/bip.360291415. [DOI] [PubMed] [Google Scholar]
  5. Chan W. W. Combination plots as graphical tools in the study of enzyme inhibition. Biochem J. 1995 Nov 1;311(Pt 3):981–985. doi: 10.1042/bj3110981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chase T., Jr, Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry. 1969 May;8(5):2212–2224. doi: 10.1021/bi00833a063. [DOI] [PubMed] [Google Scholar]
  7. Cole P. W., Murakami K., Inagami T. Specificity and mechanism of clostripain catalysis. Biochemistry. 1971 Nov;10(23):4246–4252. doi: 10.1021/bi00799a015. [DOI] [PubMed] [Google Scholar]
  8. DIXON M. The determination of enzyme inhibitor constants. Biochem J. 1953 Aug;55(1):170–171. doi: 10.1042/bj0550170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davis M. E., Madura J. D., Sines J., Luty B. A., Allison S. A., McCammon J. A. Diffusion-controlled enzymatic reactions. Methods Enzymol. 1991;202:473–497. doi: 10.1016/0076-6879(91)02024-4. [DOI] [PubMed] [Google Scholar]
  10. Drenth J., Kalk K. H., Swen H. M. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry. 1976 Aug 24;15(17):3731–3738. doi: 10.1021/bi00662a014. [DOI] [PubMed] [Google Scholar]
  11. Emöd I., Keil B. Five Sepharose-bound ligands for the chromatographic purification of Clostridium collagenase and clostripain. FEBS Lett. 1977 May 1;77(1):51–56. doi: 10.1016/0014-5793(77)80191-9. [DOI] [PubMed] [Google Scholar]
  12. Evnin L. B., Vásquez J. R., Craik C. S. Substrate specificity of trypsin investigated by using a genetic selection. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6659–6663. doi: 10.1073/pnas.87.17.6659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Foucault G., Seydoux F., Yon J. Comparative kinetic properties of alpha, beta and psi forms of trypsin. Eur J Biochem. 1974 Sep 1;47(2):295–302. doi: 10.1111/j.1432-1033.1974.tb03693.x. [DOI] [PubMed] [Google Scholar]
  14. Guenot J., Kollman P. A. Molecular dynamics studies of a DNA-binding protein: 2. An evaluation of implicit and explicit solvent models for the molecular dynamics simulation of the Escherichia coli trp repressor. Protein Sci. 1992 Sep;1(9):1185–1205. doi: 10.1002/pro.5560010912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartley B. S., Brown J. R., Kauffman D. L., Smillie L. B. Evolutionary similarities between pancreatic proteolytic enzymes. Nature. 1965 Sep 11;207(5002):1157–1159. doi: 10.1038/2071157a0. [DOI] [PubMed] [Google Scholar]
  16. MARES-GUIA M., SHAW E. STUDIES ON THE ACTIVE CENTER OF TRYPSIN. THE BINDING OF AMIDINES AND GUANIDINES AS MODELS OF THE SUBSTRATE SIDE CHAIN. J Biol Chem. 1965 Apr;240:1579–1585. [PubMed] [Google Scholar]
  17. Malthouse J. P., Brocklehurst K. Preparation of fully active ficin from Ficus glabrata by covalent chromatography and characterization of its active centre by using 2,2'-depyridyl disulphide as a reactivity probe. Biochem J. 1976 Nov;159(2):221–234. doi: 10.1042/bj1590221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Perona J. J., Tsu C. A., McGrath M. E., Craik C. S., Fletterick R. J. Relocating a negative charge in the binding pocket of trypsin. J Mol Biol. 1993 Apr 5;230(3):934–949. doi: 10.1006/jmbi.1993.1211. [DOI] [PubMed] [Google Scholar]
  19. Pike R., McGraw W., Potempa J., Travis J. Lysine- and arginine-specific proteinases from Porphyromonas gingivalis. Isolation, characterization, and evidence for the existence of complexes with hemagglutinins. J Biol Chem. 1994 Jan 7;269(1):406–411. [PubMed] [Google Scholar]
  20. Schroeder D. D., Shaw E. Chromatography of trypsin and its derivatives. Characterization of a new active form of bovine trypsin. J Biol Chem. 1968 Jun 10;243(11):2943–2949. [PubMed] [Google Scholar]
  21. Shah H. N., Gharbia S. E., Kowlessur D., Wilkie E., Brocklehurst K. Isolation and characterization of gingivain, a cysteine proteinase from Porphyromonas gingivalis strain W83. Biochem Soc Trans. 1990 Aug;18(4):578–579. doi: 10.1042/bst0180578. [DOI] [PubMed] [Google Scholar]
  22. Shaw E. Cysteinyl proteinases and their selective inactivation. Adv Enzymol Relat Areas Mol Biol. 1990;63:271–347. doi: 10.1002/9780470123096.ch5. [DOI] [PubMed] [Google Scholar]
  23. Smith R. L., Shaw E. Pseudotrypsin. A modified bovine trypsin produced by limited autodigestion. J Biol Chem. 1969 Sep 10;244(17):4704–4712. [PubMed] [Google Scholar]
  24. Soman K., Yang A. S., Honig B., Fletterick R. Electrical potentials in trypsin isozymes. Biochemistry. 1989 Dec 26;28(26):9918–9926. doi: 10.1021/bi00452a007. [DOI] [PubMed] [Google Scholar]
  25. Sreedharan S. K., Patel H., Smith S., Gharbia S. E., Shah H. N., Brocklehurst K. Additional evidence for the cysteine proteinase nature of gingivain the extracellular proteinase of Porphyromonas gingivalis. Biochem Soc Trans. 1993 May;21(2):218S–218S. doi: 10.1042/bst021218s. [DOI] [PubMed] [Google Scholar]
  26. Sreedharan S. K., Shah H. N., Brocklehurst K. trans-epoxysuccinyl-L-leucylamido (4-guanidino)-butane (E-64) inhibits trypsin-catalysed hydrolysis possibly by binding in the S1-subsite. Biochem Soc Trans. 1994 May;22(2):212S–212S. doi: 10.1042/bst022212s. [DOI] [PubMed] [Google Scholar]
  27. Tanner J. J., Smith P. E., Krause K. L. Molecular dynamics simulations and rigid body (TLS) analysis of aspartate carbamoyltransferase: evidence for an uncoupled R state. Protein Sci. 1993 Jun;2(6):927–935. doi: 10.1002/pro.5560020606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Varughese K. I., Ahmed F. R., Carey P. R., Hasnain S., Huber C. P., Storer A. C. Crystal structure of a papain-E-64 complex. Biochemistry. 1989 Feb 7;28(3):1330–1332. doi: 10.1021/bi00429a058. [DOI] [PubMed] [Google Scholar]
  29. Varughese K. I., Su Y., Cromwell D., Hasnain S., Xuong N. H. Crystal structure of an actinidin-E-64 complex. Biochemistry. 1992 Jun 9;31(22):5172–5176. doi: 10.1021/bi00137a012. [DOI] [PubMed] [Google Scholar]
  30. Yamamoto D., Ohishi H., Ishida T., Inoue M., Sumiya S., Kitamura K. Molecular dynamics simulation of papain-E-64 (N-[N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl]agmatine) complex. Chem Pharm Bull (Tokyo) 1990 Sep;38(9):2339–2343. doi: 10.1248/cpb.38.2339. [DOI] [PubMed] [Google Scholar]

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