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. 1991 May 1;275(Pt 3):751–757. doi: 10.1042/bj2750751

A model to explain the pH-dependent specificity of cathepsin B-catalysed hydrolyses.

H E Khouri 1, C Plouffe 1, S Hasnain 1, T Hirama 1, A C Storer 1, R Ménard 1
PMCID: PMC1150117  PMID: 2039451

Abstract

1. Three synthetic substrates of cathepsin B (EC 3.4.22.1) with various amino acid residues at the P2 position (Cbz-Phe-Arg-NH-Mec, Cbz-Arg-Arg-NH-Mec and Cbz-Cit-Arg-NH-Mec, where Cbz represents benzyloxycarbonyl and NH-Mec represents 4-methylcoumarin-7-ylamide) were used to investigate the pH-dependency of cathepsin B-catalysed hydrolyses and to obtain information on the nature of enzyme-substrate interactions. 2. Non-linear-regression analysis of pH-activity profiles for these substrates indicates that at least four ionizable groups on cathepsin B with pKa values of 3.3, 4.55, 5.46 and greater than 7.3 can affect the rate of substrate hydrolysis. 3. Ionization of the residue with a pKa of 5.46 has a strong effect on activity towards the substrate with an arginine in P2 (8.4-fold increase in activity) but has only a moderate effect on the rate of hydrolysis with Cbz-Cit-Arg-NH-Mec (2.3-fold increase in activity) and virtually no effect with Cbz-Phe-Arg-NH-Mec. The kinetic data are consistent with this group being an acid residue with a side chain able to interact with the side chains of an arginine or a citrulline in the P2 position of a substrate. Amino acid sequence alignment and model building with the related enzyme papain (EC 3.4.22.2) suggest that Glu-245 of cathepsin B is a likely candidate. The relative importance of electrostatic and hydrophobic interactions in the S2 subsite of cathepsin B is discussed. 4. For all three substrates, activity appears after ionization of a group with a pKa of 3.3, believed to be the active-site Cys-29 of cathepsin B. The identity of the groups with pKa values of 4.55 and greater than 7.3 remains unknown.

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Selected References

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  1. Aronson N. N., Jr, Barrett A. J. The specificity of cathepsin B. Hydrolysis of glucagon at the C-terminus by a peptidyldipeptidase mechanism. Biochem J. 1978 Jun 1;171(3):759–765. doi: 10.1042/bj1710759. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bajkowski A. S., Frankfater A. The pH dependency of bovine spleen cathepsin B-catalyzed transfer of N alpha-benzyloxycarbonyl-L-lysine from p-nitrophenol to water and dipeptide nucleophiles. Comparisons with papain. J Biol Chem. 1983 Feb 10;258(3):1650–1655. [PubMed] [Google Scholar]
  3. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Bond J. S., Barrett A. J. Degradation of fructose-1,6-bisphosphate aldolase by cathepsin B. Biochem J. 1980 Jul 1;189(1):17–25. doi: 10.1042/bj1890017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brocklehurst K., Willenbrock S. J., Salih E. Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism. Biochem J. 1983 Jun 1;211(3):701–708. doi: 10.1042/bj2110701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dufour E. Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin. Biochimie. 1988 Oct;70(10):1335–1342. doi: 10.1016/0300-9084(88)90004-1. [DOI] [PubMed] [Google Scholar]
  9. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  10. Gray P. J., Duggleby R. G. Analysis of kinetic data for irreversible enzyme inhibition. Biochem J. 1989 Jan 15;257(2):419–424. doi: 10.1042/bj2570419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hirao T., Hara K., Takahashi K. Purification and characterization of cathepsin B from monkey skeletal muscle. J Biochem. 1984 Mar;95(3):871–879. doi: 10.1093/oxfordjournals.jbchem.a134680. [DOI] [PubMed] [Google Scholar]
  12. Kamphuis I. G., Drenth J., Baker E. N. Thiol proteases. Comparative studies based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and H, and stem bromelain. J Mol Biol. 1985 Mar 20;182(2):317–329. doi: 10.1016/0022-2836(85)90348-1. [DOI] [PubMed] [Google Scholar]
  13. Katunuma N., Towatari T., Tamai M., Hanada K. Use of new synthetic substrates for assays of cathepsin L and cathepsin B. J Biochem. 1983 Apr;93(4):1129–1135. doi: 10.1093/oxfordjournals.jbchem.a134238. [DOI] [PubMed] [Google Scholar]
  14. Knight C. G. Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide to a study of the inhibition by leupeptin. Biochem J. 1980 Sep 1;189(3):447–453. doi: 10.1042/bj1890447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mason R. W. Interaction of lysosomal cysteine proteinases with alpha 2-macroglobulin: conclusive evidence for the endopeptidase activities of cathepsins B and H. Arch Biochem Biophys. 1989 Sep;273(2):367–374. doi: 10.1016/0003-9861(89)90495-5. [DOI] [PubMed] [Google Scholar]
  16. McKay M. J., Offermann M. K., Barrett A. J., Bond J. S. Action of human liver cathepsin B on the oxidized insulin B chain. Biochem J. 1983 Aug 1;213(2):467–471. doi: 10.1042/bj2130467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ménard R., Khouri H. E., Plouffe C., Dupras R., Ripoll D., Vernet T., Tessier D. C., Lalberté F., Thomas D. Y., Storer A. C. A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain. Biochemistry. 1990 Jul 17;29(28):6706–6713. doi: 10.1021/bi00480a021. [DOI] [PubMed] [Google Scholar]
  18. Polgár L., Csoma C. Dissociation of ionizing groups in the binding cleft inversely controls the endo- and exopeptidase activities of cathepsin B. J Biol Chem. 1987 Oct 25;262(30):14448–14453. [PubMed] [Google Scholar]
  19. Rich D. H., Brown M. A., Barrett A. J. Purification of cathepsin B by a new form of affinity chromatography. Biochem J. 1986 May 1;235(3):731–734. doi: 10.1042/bj2350731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Willenbrock F., Brocklehurst K. A general framework of cysteine-proteinase mechanism deduced from studies on enzymes with structurally different analogous catalytic-site residues Asp-158 and -161 (papain and actinidin), Gly-196 (cathepsin B) and Asn-165 (cathepsin H). Kinetic studies up to pH 8 of the hydrolysis of N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide catalysed by cathepsin B and of L-arginine 2-naphthylamide catalysed by cathepsin H. Biochem J. 1985 Apr 15;227(2):521–528. doi: 10.1042/bj2270521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Willenbrock F., Brocklehurst K. Natural structural variation in enzymes as a tool in the study of mechanism exemplified by a comparison of the catalytic-site structure and characteristics of cathepsin B and papain. pH-dependent kinetics of the reactions of cathepsin B from bovine spleen and from rat liver with a thiol-specific two-protonic-state probe (2,2'-dipyridyl disulphide) and with a specific synthetic substrate (N-alpha-benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide). Biochem J. 1984 Sep 15;222(3):805–814. doi: 10.1042/bj2220805. [DOI] [PMC free article] [PubMed] [Google Scholar]

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