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Biochem J. 2002 Feb 1; 361(Pt 3): 613–619.
PMCID: PMC1222344

S2' substrate specificity and the role of His110 and His111 in the exopeptidase activity of human cathepsin B.


The ability of the lysosomal cysteine protease cathepsin B to function as a peptidyldipeptidase (removing C-terminal dipeptides) has been attributed to the presence of two histidine residues (His(110) and His(111)) present in the occluding loop, an extra peptide segment located in the primed side of the active-site cleft. Whereas His(111) is unpaired, His(110) is present as an ion pair with Asp(22) on the main body of the protease. This ion pair appears to act as a latch to hold the loop in a closed position. The exopeptidase activity of cathepsin B, examined using quenched fluorescence substrates, was shown to have a 20-fold preference for aromatic side chains in the P2' position relative to glutamic acid as the least favourable residue. Site-directed mutagenesis demonstrated that His(111) makes a positive 10-fold contribution to the exopeptidase activity, whereas His(110) is critical for this action with the Asp(22)-His(110) ion pair stabilizing the electrostatic interaction by a maximum of 13.9 kJ/mol (3.3 kcal/mol). These studies showed that cathepsin B is optimized to act as an exopeptidase, cleaving dipeptides from protein substrates in a successive manner, because of its relaxed specificity in P2' and its other subsites.

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Selected References

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  • Schechter I, Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. [PubMed]
  • Mort JS, Buttle DJ. Cathepsin B. Int J Biochem Cell Biol. 1997 May;29(5):715–720. [PubMed]
  • Halangk W, Lerch MM, Brandt-Nedelev B, Roth W, Ruthenbuerger M, Reinheckel T, Domschke W, Lippert H, Peters C, Deussing J. Role of cathepsin B in intracellular trypsinogen activation and the onset of acute pancreatitis. J Clin Invest. 2000 Sep;106(6):773–781. [PMC free article] [PubMed]
  • Mort JS, Recklies AD, Poole AR. Extracellular presence of the lysosomal proteinase cathepsin B in rheumatoid synovium and its activity at neutral pH. Arthritis Rheum. 1984 May;27(5):509–515. [PubMed]
  • Sloane BF. Cathepsin B and cystatins: evidence for a role in cancer progression. Semin Cancer Biol. 1990 Apr;1(2):137–152. [PubMed]
  • Guicciardi ME, Deussing J, Miyoshi H, Bronk SF, Svingen PA, Peters C, Kaufmann SH, Gores GJ. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest. 2000 Nov;106(9):1127–1137. [PMC free article] [PubMed]
  • Nägler DK, Storer AC, Portaro FC, Carmona E, Juliano L, Ménard R. Major increase in endopeptidase activity of human cathepsin B upon removal of occluding loop contacts. Biochemistry. 1997 Oct 14;36(41):12608–12615. [PubMed]
  • Barrett AJ, Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. [PubMed]
  • Musil D, Zucic D, Turk D, Engh RA, Mayr I, Huber R, Popovic T, Turk V, Towatari T, Katunuma N, et al. The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity. EMBO J. 1991 Sep;10(9):2321–2330. [PMC free article] [PubMed]
  • Cygler M, Sivaraman J, Grochulski P, Coulombe R, Storer AC, Mort JS. Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion. Structure. 1996 Apr 15;4(4):405–416. [PubMed]
  • Podobnik M, Kuhelj R, Turk V, Turk D. Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen. J Mol Biol. 1997 Sep 5;271(5):774–788. [PubMed]
  • Turk D, Podobnik M, Popovic T, Katunuma N, Bode W, Huber R, Turk V. Crystal structure of cathepsin B inhibited with CA030 at 2.0-A resolution: A basis for the design of specific epoxysuccinyl inhibitors. Biochemistry. 1995 Apr 11;34(14):4791–4797. [PubMed]
  • Yamamoto A, Hara T, Tomoo K, Ishida T, Fujii T, Hata Y, Murata M, Kitamura K. Binding mode of CA074, a specific irreversible inhibitor, to bovine cathepsin B as determined by X-ray crystal analysis of the complex. J Biochem. 1997 May;121(5):974–977. [PubMed]
  • Illy C, Quraishi O, Wang J, Purisima E, Vernet T, Mort JS. Role of the occluding loop in cathepsin B activity. J Biol Chem. 1997 Jan 10;272(2):1197–1202. [PubMed]
  • Quraishi O, Nägler DK, Fox T, Sivaraman J, Cygler M, Mort JS, Storer AC. The occluding loop in cathepsin B defines the pH dependence of inhibition by its propeptide. Biochemistry. 1999 Apr 20;38(16):5017–5023. [PubMed]
  • Meldal M, Breddam K. Anthranilamide and nitrotyrosine as a donor-acceptor pair in internally quenched fluorescent substrates for endopeptidases: multicolumn peptide synthesis of enzyme substrates for subtilisin Carlsberg and pepsin. Anal Biochem. 1991 May 15;195(1):141–147. [PubMed]
  • Kunkel TA, Roberts JD, Zakour RA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. [PubMed]
  • Mach L, Schwihla H, Stüwe K, Rowan AD, Mort JS, Glössl J. Activation of procathepsin B in human hepatoma cells: the conversion into the mature enzyme relies on the action of cathepsin B itself. Biochem J. 1993 Jul 15;293(Pt 2):437–442. [PMC free article] [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Rawlings ND, Barrett AJ. FLUSYS: a software package for the collection and analysis of kinetic and scanning data from Perkin-Elmer fluorimeters. Comput Appl Biosci. 1990 Apr;6(2):118–119. [PubMed]
  • Taylor S, Ninjoor V, Dowd DM, Tappel AL. Cathepsin B2 measurement by sensitive fluorometric ammonia analysis. Anal Biochem. 1974 Jul;60(1):153–162. [PubMed]
  • Nägler DK, Tam W, Storer AC, Krupa JC, Mort JS, Ménard R. Interdependency of sequence and positional specificities for cysteine proteases of the papain family. Biochemistry. 1999 Apr 13;38(15):4868–4874. [PubMed]
  • Kirschke H, Locnikar P, Turk V. Species variations amongst lysosomal cysteine proteinases. FEBS Lett. 1984 Aug 20;174(1):123–127. [PubMed]
  • Ménard R, Carmona E, Plouffe C, Brömme D, Konishi Y, Lefebvre J, Storer AC. The specificity of the S1' subsite of cysteine proteases. FEBS Lett. 1993 Aug 9;328(1-2):107–110. [PubMed]
  • Therrien C, Lachance P, Sulea T, Purisima EO, Qi H, Ziomek E, Alvarez-Hernandez A, Roush WR, Ménard R. Cathepsins X and B can be differentiated through their respective mono- and dipeptidyl carboxypeptidase activities. Biochemistry. 2001 Mar 6;40(9):2702–2711. [PubMed]
  • Wilkinson AJ, Fersht AR, Blow DM, Winter G. Site-directed mutagenesis as a probe of enzyme structure and catalysis: tyrosyl-tRNA synthetase cysteine-35 to glycine-35 mutation. Biochemistry. 1983 Jul 19;22(15):3581–3586. [PubMed]
  • Koga H, Yamada H, Nishimura Y, Kato K, Imoto T. Multiple proteolytic action of rat liver cathepsin B: specificities and pH-dependences of the endo- and exopeptidase activities. J Biochem. 1991 Aug;110(2):179–188. [PubMed]
  • Takahashi T, Dehdarani AH, Yonezawa S, Tang J. Porcine spleen cathepsin B is an exopeptidase. J Biol Chem. 1986 Jul 15;261(20):9375–9381. [PubMed]
  • Rowan AD, Feng R, Konishi Y, Mort JS. Demonstration by electrospray mass spectrometry that the peptidyldipeptidase activity of cathepsin B is capable of rat cathepsin B C-terminal processing. Biochem J. 1993 Sep 15;294(Pt 3):923–927. [PMC free article] [PubMed]
  • Berti PJ, Faerman CH, Storer AC. Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites. Biochemistry. 1991 Feb 5;30(5):1394–1402. [PubMed]
  • Turk B, Turk D, Turk V. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta. 2000 Mar 7;1477(1-2):98–111. [PubMed]
  • Guncar G, Podobnik M, Pungercar J, Strukelj B, Turk V, Turk D. Crystal structure of porcine cathepsin H determined at 2.1 A resolution: location of the mini-chain C-terminal carboxyl group defines cathepsin H aminopeptidase function. Structure. 1998 Jan 15;6(1):51–61. [PubMed]
  • Cigić B, Krizaj I, Kralj B, Turk V, Pain RH. Stoichiometry and heterogeneity of the pro-region chain in tetrameric human cathepsin C. Biochim Biophys Acta. 1998 Jan 15;1382(1):143–150. [PubMed]
  • Sivaraman J, Nägler DK, Zhang R, Ménard R, Cygler M. Crystal structure of human procathepsin X: a cysteine protease with the proregion covalently linked to the active site cysteine. J Mol Biol. 2000 Jan 28;295(4):939–951. [PubMed]
  • Guncar G, Klemencic I, Turk B, Turk V, Karaoglanovic-Carmona A, Juliano L, Turk D. Crystal structure of cathepsin X: a flip-flop of the ring of His23 allows carboxy-monopeptidase and carboxy-dipeptidase activity of the protease. Structure. 2000 Mar 15;8(3):305–313. [PubMed]
  • Nycander M, Estrada S, Mort JS, Abrahamson M, Björk I. Two-step mechanism of inhibition of cathepsin B by cystatin C due to displacement of the proteinase occluding loop. FEBS Lett. 1998 Jan 23;422(1):61–64. [PubMed]
  • Pavlova A, Krupa JC, Mort JS, Abrahamson M, Björk I. Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration By release of loop anchoring through mutation of his110. FEBS Lett. 2000 Dec 29;487(2):156–160. [PubMed]
  • Taralp A, Kaplan H, Sytwu II, Vlattas I, Bohacek R, Knap AK, Hirama T, Huber CP, Hasnain S. Characterization of the S3 subsite specificity of cathepsin B. J Biol Chem. 1995 Jul 28;270(30):18036–18043. [PubMed]
  • Brömme D, Bonneau PR, Lachance P, Storer AC. Engineering the S2 subsite specificity of human cathepsin S to a cathepsin L- and cathepsin B-like specificity. J Biol Chem. 1994 Dec 2;269(48):30238–30242. [PubMed]
  • Brömme D, Okamoto K, Wang BB, Biroc S. Human cathepsin O2, a matrix protein-degrading cysteine protease expressed in osteoclasts. Functional expression of human cathepsin O2 in Spodoptera frugiperda and characterization of the enzyme. J Biol Chem. 1996 Jan 26;271(4):2126–2132. [PubMed]
  • Brömme D, Li Z, Barnes M, Mehler E. Human cathepsin V functional expression, tissue distribution, electrostatic surface potential, enzymatic characterization, and chromosomal localization. Biochemistry. 1999 Feb 23;38(8):2377–2385. [PubMed]
  • Hasnain S, Hirama T, Huber CP, Mason P, Mort JS. Characterization of cathepsin B specificity by site-directed mutagenesis. Importance of Glu245 in the S2-P2 specificity for arginine and its role in transition state stabilization. J Biol Chem. 1993 Jan 5;268(1):235–240. [PubMed]
  • Aronson NN, Jr, Barrett AJ. 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. [PMC free article] [PubMed]
  • Mort JS, Magny MC, Lee ER. Cathepsin B: an alternative protease for the generation of an aggrecan 'metalloproteinase' cleavage neoepitope. Biochem J. 1998 Nov 1;335(Pt 3):491–494. [PMC free article] [PubMed]
  • Authier F, Métioui M, Bell AW, Mort JS. Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B. J Biol Chem. 1999 Nov 19;274(47):33723–33731. [PubMed]

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