Cysteine protease

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Cysteine Peptidase
Papain enzyme.png
Crystal structure of the cysteine peptidase papain in complex with its covalent inhibitor E-64. Rendered from PDB 1PE6
Symbol Peptidase_C1
Pfam PF00112
Pfam clan CL0125
InterPro IPR000668
SCOP 1aec
OPM superfamily 420
OPM protein 1m6d

Cysteine proteases, also known as thiol proteases, are enzymes that degrade proteins. These proteases share a common catalytic mechanism that involves a nucleophilic cysteine thiol in a catalytic triad or dyad.

Cysteine proteases are commonly encountered in fruits including the papaya, pineapple, fig and kiwifruit. The proportion of protease tends to be higher when the fruit is unripe. In fact, dozens of latices of different plant families are known to contain cysteine proteases.[1] Cysteine proteases are used as an ingredient in meat tenderizers.


The MEROPS protease classification system counts 14 superfamilies plus several currently unassigned families (as of 2013) each containing many families. Each superfamily uses the catalytic triad or dyad in a different protein fold and so represent convergent evolution of the catalytic mechanism.

For superfamilies, P = superfamily containing a mixture of nucleophile class families, C = purely cysteine proteases. superfamily. Within each superfamily, families are designated by their catalytic nucleophile (C = cysteine proteases).

Superfamily Families Examples
PA C3, C4, C24, C30, C37, C62, C74, C99 TEV protease (Tobacco etch virus)[2]
PB C44, C45, C59, C69, C89, C95 amidophosphoribosyltransferase precursor (Homo sapiens)[3]
PC C26, C56 gamma-glutamyl hydrolase (Rattus norvegicus)[4]
PD C46 hedgehog protein (Drosophila melanogaster)[5]
PE P1 DmpA aminopeptidase (Ochrobactrum anthropi)[6]
CA C1, C2, C6, C10, C12, C16, C19, C28, C31, C32, C33, C39, C47, C51, C54, C58, C64,

C65, C66, C67, C70, C71, C76, C78, C83, C85, C86, C87, C93, C96, C98, C101

papain (Carica papaya),[7] bromelain (Ananas comosus), cathepsin K (liverwort)[8] and calpain (Homo sapiens)[9]
CD C11, C13, C14, C25, C50, C80, C84 caspase-1 (Rattus norvegicus)[10] and separase (Saccharomyces cerevisiae)[11]
CE C5, C48, C55, C57, C63, C79 adenain (human adenovirus type 2)[12]
CF C15 pyroglutamyl-peptidase I (Bacillus amyloliquefaciens)[13]
CL C60, C82 sortase A (Staphylococcus aureus)[14]
CM C18 hepatitis C virus peptidase 2 (hepatitis C virus)[15]
CN C9 sindbis virus-type nsP2 peptidase (sindbis virus)[16]
CO C40 dipeptidyl-peptidase VI (Lysinibacillus sphaericus)[17]
CP C97 DeSI-1 peptidase (Mus musculus)[18]
unassigned C7, C8, C21, C23, C27, C36, C42, C53, C75

Catalytic mechanism[edit]

Reaction mechanism of the cysteine protease mediated cleavage of a peptide bond.
See also: catalytic triad

The first step in the reaction mechanism by which cysteine proteases catalyze the hydrolysis of peptide bonds is deprotonation of a thiol in the enzyme's active site by an adjacent amino acid with a basic side chain, usually a histidine residue. The next step is nucleophilic attack by the deprotonated cysteine's anionic sulfur on the substrate carbonyl carbon. In this step, a fragment of the substrate is released with an amine terminus, the histidine residue in the protease is restored to its deprotonated form, and a thioester intermediate linking the new carboxy-terminus of the substrate to the cysteine thiol is formed. Therefore they are also sometimes referred to as thiol proteases. The thioester bond is subsequently hydrolyzed to generate a carboxylic acid moiety on the remaining substrate fragment, while regenerating the free enzyme.

Biological importance[edit]

Cysteine proteases play multi-faceted roles, virtually in every aspect of physiology and development. In plants they are important in growth and development and in accumulation and mobilization of storage proteins such as in seeds. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses.[19] In humans and other animals, they are responsible for senescence and apoptosis (programmed cell death), MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development. The ability of macrophages and other cells to mobilize elastolytic cysteine proteases to their surfaces under specialized conditions may also lead to accelerated collagen and elastin degradation at sites of inflammation in diseases such as atherosclerosis and emphysema.[20] Several viruses (e.g. polio, hepatitis C) express their entire genome as a singe massive polyprotein and use a protease to cleave it into functional units (e.g. Tobacco Etch Virus protease).


Proteases are usually synthesized as large precursor proteins called zymogens, such as the serine protease precursors trypsinogen and chymotrypsinogen, and the aspartic protease precursor pepsinogen. The protease is activated by removal of an inhibitory segment or protein. Activation occurs once the protease is delivered to a specific intracellular compartment (e.g. lysosome) or extracellular environment (e.g. stomach). This system prevents the cell that produces the protease from being damaged by it.

Protease inhibitors are usually proteins with domains that enter or block a protease active site to prevent substrate access. In competitive inhibition, the inhibitor binds to the active site, thus preventing enzyme-substrate interaction. In non-competitive inhibition, the inhibitor binds to an allosteric site, which alters the active site and makes it inaccessible to the substrate.

Examples of protease inhibitors include:


Potential pharmaceuticals[edit]

Currently there is no widespread use of cysteine proteases as approved and effective anthelmintics but research into the subject is a promising field of study. Plant cysteine proteases isolated from these plants have been found to have high proteolytic activities that are known to digest nematode cuticles, with very low toxicity.[21] Successful results have been reported against nematodes such as Heligmosomoides bakeri, Trichinella spiralis, Nippostrongylus brasiliensis, Trichuris muris, and Ancylostoma ceylanicum; the tapeworm Rodentolepis microstoma, and the porcine acanthocephalan parasite Macracanthorynchus hirundinaceus.[22] A useful property of cysteine proteases is the resistance to acid digestion, allowing possible oral administration. They provide an alternative mechanism of action to current anthelmintics and the development of resistance is thought to be unlikely because it would require a complete change of structure of the helminth cuticle.

In several traditional medicines, the fruits or latex of the papaya, pineapple and fig are widely used for treatment of intestinal worm infections both in humans and livestock.


Cysteine proteases are used as feed additives for livestock to improve the digestibility of protein. They hydrolyse complex proteins into simple amino acids through gut.[citation needed]

See also[edit]


  1. ^ Domsalla A, Melzig MF (June 2008). "Occurrence and properties of proteases in plant latices". Planta Med. 74 (7): 699–711. doi:10.1055/s-2008-1074530. PMID 18496785. 
  2. ^ Phan J, Zdanov A, Evdokimov AG, Tropea JE, Peters HK, Kapust RB, Li M, Wlodawer A, Waugh DS (December 2002). "Structural basis for the substrate specificity of tobacco etch virus protease". J. Biol. Chem. 277 (52): 50564–72. doi:10.1074/jbc.M207224200. PMID 12377789. 
  3. ^ Iwahana H, Oka J, Mizusawa N et al. (1993). "Molecular cloning of human amidophosphoribosyltransferase.". Biochem. Biophys. Res. Commun. 190 (1): 192–200. doi:10.1006/bbrc.1993.1030. PMID 8380692. 
  4. ^ Galivan, J; Ryan, T; Rhee, M; Yao, R; Chave, K (1999). "Glutamyl hydrolase: Properties and pharmacologic impact". Seminars in oncology 26 (2 Suppl 6): 33–7. PMID 10598552.  edit
  5. ^ Ingham, P. W.; Nakano, Y; Seger, C (2011). "Mechanisms and functions of Hedgehog signalling across the metazoa". Nature Reviews Genetics 12 (6): 393–406. doi:10.1038/nrg2984. PMID 21502959.  edit
  6. ^ Geueke, B; Heck, T; Limbach, M; Nesatyy, V; Seebach, D; Kohler, H. P. (2006). "Bacterial beta-peptidyl aminopeptidases with unique substrate specificities for beta-oligopeptides and mixed beta,alpha-oligopeptides". FEBS Journal 273 (23): 5261–72. doi:10.1111/j.1742-4658.2006.05519.x. PMID 17064315.  edit
  7. ^ Mitchel, R. E.; Chaiken, I. M.; Smith, E. L. (1970). "The complete amino acid sequence of papain. Additions and corrections". The Journal of biological chemistry 245 (14): 3485–92. PMID 5470818.  edit
  8. ^ Sierocka, I; Kozlowski, L. P.; Bujnicki, J. M.; Jarmolowski, A; Szweykowska-Kulinska, Z (2014). "Female-specific gene expression in dioecious liverwort Pellia endiviifolia is developmentally regulated and connected to archegonia production". BMC Plant Biology 14: 168. doi:10.1186/1471-2229-14-168. PMC 4074843. PMID 24939387.  edit
  9. ^ Sorimachi, H; Ohmi, S; Emori, Y; Kawasaki, H; Saido, T. C.; Ohno, S; Minami, Y; Suzuki, K (1990). "A novel member of the calcium-dependent cysteine protease family". Biological chemistry Hoppe-Seyler. 371 Suppl: 171–6. PMID 2400579.  edit
  10. ^ Black, R. A.; Kronheim, S. R.; Merriam, J. E.; March, C. J.; Hopp, T. P. (1989). "A pre-aspartate-specific protease from human leukocytes that cleaves pro-interleukin-1 beta". The Journal of biological chemistry 264 (10): 5323–6. PMID 2784432.  edit
  11. ^ Waizenegger, I; Giménez-Abián, J. F.; Wernic, D; Peters, J. M. (2002). "Regulation of human separase by securin binding and autocleavage". Current biology : CB 12 (16): 1368–78. doi:10.1016/s0960-9822(02)01073-4. PMID 12194817.  edit
  12. ^ Webster, A; Hay, R. T.; Kemp, G (1993). "The adenovirus protease is activated by a virus-coded disulphide-linked peptide". Cell 72 (1): 97–104. doi:10.1016/0092-8674(93)90053-s. PMID 8422686.  edit
  13. ^ Tsuru, D., Nakamura, K., Yoshimoto, T. and Fujiwara, K. (1984). "Pyroglutamyl-peptidase from Bacillus amyloliquefaciens. An improved purification method and some properties of the enzyme". Biochim. Biophys. Acta 791: 117–122. doi:10.1016/0167-4838(84)90001-3. 
  14. ^ Ton-That, H., Liu, G., Mazmanian, S.K., Faull, K.F. and Schneewind, O. (1999). "Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif". Proc. Natl. Acad. Sci. USA 96: 12424–12429. doi:10.1073/pnas.96.22.12424. PMID 10535938. 
  15. ^ Miyamura, T; Suzuki, T; Mathuura, Y (1993). "Structural proteins of hepatitis C virus". Uirusu 43 (2): 275–83. PMID 7510088.  edit
  16. ^ Strauss, E. G.; De Groot, R. J.; Levinson, R; Strauss, J. H. (1992). "Identification of the active site residues in the nsP2 proteinase of Sindbis virus". Virology 191 (2): 932–40. doi:10.1016/0042-6822(92)90268-t. PMID 1448929.  edit
  17. ^ Hourdou, M. L.; Duez, C; Joris, B; Vacheron, M. J.; Guinand, M; Michel, G; Ghuysen, J. M. (1992). "Cloning and nucleotide sequence of the gene encoding the gamma-D-glutamyl-L-diamino acid endopeptidase II of Bacillus sphaericus". FEMS microbiology letters 70 (2): 165–70. PMID 1587462.  edit
  18. ^ Suh, H. Y.; Kim, J. H.; Woo, J. S.; Ku, B; Shin, E. J.; Yun, Y; Oh, B. H. (2012). "Crystal structure of DeSI-1, a novel deSUMOylase belonging to a putative isopeptidase superfamily". Proteins: Structure, Function, and Bioinformatics 80 (8): 2099–104. doi:10.1002/prot.24093. PMID 22498933.  edit
  19. ^ Grudkowska M, Zagdańska B (2004). "Multifunctional role of plant cysteine proteinases" (PDF). Acta Biochim. Pol. 51 (3): 609–24. PMID 15448724. 
  20. ^ Chapman HA, Riese RJ, Shi GP (1997). "Emerging roles for cysteine proteases in human biology". Annu. Rev. Physiol. 59: 63–88. doi:10.1146/annurev.physiol.59.1.63. PMID 9074757. 
  21. ^ Stepek G, Behnke JM, Buttle DJ, Duce IR (July 2004). "Natural plant cysteine proteinases as anthelmintics?". Trends Parasitol. 20 (7): 322–7. doi:10.1016/ PMID 15193563. 
  22. ^ Behnke JM, Buttle DJ, Stepek G, Lowe A, Duce IR (2008). "Developing novel anthelmintics from plant cysteine proteinases". Parasit Vectors 1 (1): 29. doi:10.1186/1756-3305-1-29. PMC 2559997. PMID 18761736. 

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