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PROSITE documentation PDOC00126

Eukaryotic thiol (cysteine) proteases active sites


Eukaryotic thiol proteases (EC 3.4.22.-) [1] are a family of proteolytic enzymes which contain an active site cysteine. Catalysis proceeds through a thioester intermediate and is facilitated by a nearby histidine side chain; an asparagine completes the essential catalytic triad. The proteases which are currently known to belong to this family are listed below (references are only provided for recently determined sequences).

  • Vertebrate lysosomal cathepsins B (EC, H (EC, L (EC, and S (EC [2].
  • Vertebrate lysosomal dipeptidyl peptidase I (EC (also known as cathepsin C) [2].
  • Vertebrate calpains (EC (EC Calpains are intracellular calcium-activated thiol protease that contain both a N-terminal catalytic domain and a C-terminal calcium-binding domain.
  • Mammalian cathepsin K, which seems involved in osteoclastic bone resorption [3].
  • Human cathepsin O [4].
  • Bleomycin hydrolase. An enzyme that catalyzes the inactivation of the antitumor drug BLM (a glycopeptide).
  • Plant enzymes: barley aleurain (EC, EP-B1/B4; kidney bean EP-C1, rice bean SH-EP; kiwi fruit actinidin (EC; papaya latex papain (EC, chymopapain (EC, caricain (EC, and proteinase IV (EC; pea turgor-responsive protein 15A; pineapple stem bromelain (EC; rape COT44; rice oryzain α, β, and γ; tomato low-temperature induced, Arabidopsis thaliana A494, RD19A and RD21A.
  • House-dust mites allergens DerP1 and EurM1.
  • Cathepsin B-like proteinases from the worms Caenorhabditis elegans (genes gcp-1, cpr-3, cpr-4, cpr-5 and cpr-6), Schistosoma mansoni (antigen SM31) and Japonica (antigen SJ31), Haemonchus contortus (genes AC-1 and AC-2), and Ostertagia ostertagi (CP-1 and CP-3).
  • Slime mold cysteine proteinases CP1 and CP2.
  • Cruzipain from Trypanosoma cruzi and brucei.
  • Throphozoite cysteine proteinase (TCP) from various Plasmodium species.
  • Proteases from Leishmania mexicana, Theileria annulata and Theileria parva.
  • Baculoviruses cathepsin-like enzyme (v-cath).
  • Drosophila small optic lobes protein (gene sol), a neuronal protein that contains a calpain-like domain.
  • Yeast thiol protease BLH1/YCP1/LAP3.
  • Caenorhabditis elegans hypothetical protein C06G4.2, a calpain-like protein.

Two bacterial peptidases are also part of this family:

  • Aminopeptidase C from Lactococcus lactis (gene pepC) [5].
  • Thiol protease tpr from Porphyromonas gingivalis.

Three other proteins are structurally related to this family, but may have lost their proteolytic activity.

  • Soybean oil body protein P34. This protein has its active site cysteine replaced by a glycine.
  • Rat testin, a sertoli cell secretory protein highly similar to cathepsin L but with the active site cysteine is replaced by a serine. Rat testin should not be confused with mouse testin which is a LIM-domain protein (see <PDOC00382>).
  • Plasmodium falciparum serine-repeat protein (SERA), the major blood stage antigen. This protein of 111 Kd possesses a C-terminal thiol-protease-like domain [6], but the active site cysteine is replaced by a serine.

The sequences around the three active site residues are well conserved and can be used as signature patterns.


The residue in position 4 of the pattern is almost always cysteine; the only exceptions are calpains (Leu), bleomycin hydrolase (Ser) and yeast YCP1 (Ser).


The residue in position 5 of the pattern is always Gly except in papaya protease IV where it is Glu.

Consensus pattern:

[LIVMGSTAN]-{IEVK}-H-[GSACE]-[LIVM]-{GPSI}-[LIVMAT](2)-G- {SLAG}-[GSADNH] [H is the active site residue]

Sequences known to belong to this class detected by the pattern:

ALL, except for calpains, P34 and tpr.

Other sequence(s) detected in Swiss-Prot:


Consensus pattern:

[FYCH]-[WI]-[LIVT]-x-[KRQAG]-N-[ST]-W-x(3)-[FYW]-G-x(2)-G- [LFYW]-[LIVMFYG]-x-[LIVMF] [N is the active site residue]

Sequences known to belong to this class detected by the pattern:

ALL, except for calpains, bromelain, yeast BLH1, tomato low-temperature induced protease, cathepsin O, pepC and tpr.

Other sequence(s) detected in Swiss-Prot:



These proteins belong to family C1 (papain-type) and C2 (calpains) in the classification of peptidases [7,E1].

Expert(s) to contact by email:

Turk B.

Last update:

April 2006 / Patterns revised.

Technical section

PROSITE methods (with tools and information) covered by this documentation:

THIOL_PROTEASE_ASN, PS00640; Eukaryotic thiol (cysteine) proteases asparagine active site  (PATTERN)

THIOL_PROTEASE_CYS, PS00139; Eukaryotic thiol (cysteine) proteases cysteine active site  (PATTERN)

THIOL_PROTEASE_HIS, PS00639; Eukaryotic thiol (cysteine) proteases histidine active site  (PATTERN)


1AuthorsDufour E.
TitleSequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin.
SourceBiochimie 70:1335-1342(1988).
PubMed ID3148320

2AuthorsKirschke H., Barrett A.J., Rawlings N.D.
SourceProtein Prof. 2:1587-1643(1995).

3AuthorsShi G.-P., Chapman H.A., Bhairi S.M., DeLeeuw C., Reddy V.Y., Weiss S.J.
TitleMolecular cloning of human cathepsin O, a novel endoproteinase and homologue of rabbit OC2.
SourceFEBS Lett. 357:129-134(1995).
PubMed ID7805878

4AuthorsVelasco G., Ferrando A.A., Puente X.S., Sanchez L.M., Lopez-Otin C.
TitleHuman cathepsin O. Molecular cloning from a breast carcinoma, production of the active enzyme in Escherichia coli, and expression analysis in human tissues.
SourceJ. Biol. Chem. 269:27136-27142(1994).
PubMed ID7929457

5AuthorsChapot-Chartier M.P., Nardi M., Chopin M.C., Chopin A., Gripon J.C.
SourceAppl. Environ. Microbiol. 59:330-333(1993).

6AuthorsHiggins D.G., McConnell D.J., Sharp P.M.
TitleMalarial proteinase?
SourceNature 340:604-604(1989).
PubMed ID2671749

7AuthorsRawlings N.D., Barrett A.J.
TitleFamilies of cysteine peptidases.
SourceMethods Enzymol. 244:461-486(1994).
PubMed ID7845226


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