Cysteine protease Edit Wikipedia article

This article needs additional citations for verification. Please help improve this article by adding citations to reliable sources. Unsourced material may be challenged and removed. (January 2011)

Cysteine Peptidase
Crystal structure of the cysteine peptidase papain in complex with its covalent inhibtor E-64. Rendered from PDB 1PE6
Identifiers
Symbol	Peptidase_C1
Pfam	PF00112
Pfam clan	CL0125
InterPro	IPR000668
SMART	SM00645
PROSITE	PDOC00126
MEROPS	C1
SCOP	1aec
SUPERFAMILY	1aec
OPM superfamily	420
OPM protein	1m6d
Available protein structures: Pfam structures PDB RCSB PDB; PDBe PDBsum structure summary

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 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.

Reaction mechanism of the cysteine protease mediated cleavage of a peptide bond.

[edit] Classification

The cysteine proteases are classified into 16 families.

[edit] Catalytic mechanism

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.

[edit] Biological importance

Cysteine proteases play multi-faceted roles, virtually in every aspect of physiology and development, in plants such as in the growth and development, in senescence and apoptosis (programmed cell death), 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.^[2] In humans they are responsible for apoptosis, 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.^[3]

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.

[edit] Examples

Examples of enzymes that are cysteine proteases include:

[edit] Regulation

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:

• Serpins
• Stefins
• IAPs
• TIMPs

[edit] Potential pharmacological uses

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.^[4] 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.^[5] 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.

[edit] See also

• The Proteolysis Map

[edit] References

[edit] External links

• The MEROPS online database for peptidases and their inhibitors: Cysteine Peptidases
• Cysteine endopeptidases at the US National Library of Medicine Medical Subject Headings (MeSH)

Papain Edit Wikipedia article

"Accuzyme" redirects here. Accuzyme is also a DNA polymerase and PCR mix from Bioline Reagents.

Papain family cysteine protease
Papain from Carica papaya
Identifiers
Symbol	Peptidase_C1
Pfam	PF00112
InterPro	IPR000668
PROSITE	PDOC00126
SCOP	1aec
SUPERFAMILY	1aec
Available protein structures: Pfam structures PDB RCSB PDB; PDBe PDBsum structure summary

Papain, also known as papaya proteinase I, is a cysteine protease (EC 3.4.22.2) enzyme present in papaya (Carica papaya) and mountain papaya (Vasconcellea cundinamarcensis).

[edit] Papain family

Papain belongs to a family of related proteins with a wide variety of activities, including endopeptidases, aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endo-peptidase activity.^[1] Members of the papain family are widespread, found in baculovirus,^[2] eubacteria, yeast, and practically all protozoa, plants and mammals.^[1] The proteins are typically lysosomal or secreted, and proteolytic cleavage of the propeptide is required for enzyme activation, although bleomycin hydrolase is cytosolic in fungi and mammals.^[3] Papain-like cysteine proteinases are essentially synthesised as inactive proenzymes (zymogens) with N-terminal propeptide regions. The activation process of these enzymes includes the removal of propeptide regions, which serve a variety of functions in vivo and in vitro. The pro-region is required for the proper folding of the newly synthesised enzyme, the inactivation of the peptidase domain and stabilisation of the enzyme against denaturing at neutral to alkaline pH conditions. Amino acid residues within the pro-region mediate their membrane association, and play a role in the transport of the proenzyme to lysosomes. Among the most notable features of propeptides is their ability to inhibit the activity of their cognate enzymes and that certain propeptides exhibit high selectivity for inhibition of the peptidases from which they originate.^[4]

[edit] Structure

The papain precursor protein contains 345 amino acid residues,^[5] and consists of a signal sequence (1-18), a propeptide (19-133) and the mature peptide (134-345). The amino acid numbers are based on the mature peptide. The protein is stabilised by three disulfide bridges. Its three-dimensional structure consists of two distinct structural domains with a cleft between them. This cleft contains the active site, which contains a catalytic diad that has been likened to the catalytic triad of chymotrypsin. The catalytic diad is made up of the amino acids - cysteine-25 (from which it gets its classification) and histidine-159. Aspartate-158 was thought to play a role analogous to the role of aspartate in the serine protease catalytic triad, but that has since then been disproved.^[6] Nonetheless, it does appear to play a role.

[edit] Function

The mechanism by which it breaks peptide bonds involves deprotonation of Cys-25 by His-159. Asparagine-175 helps to orient the imidazole ring of His-159 to allow this deprotonation to take place. Cys-25 then performs a nucleophilic attack on the carbonyl carbon of a peptide backbone. This frees the amino terminal of the peptide, and forms a covalent acyl-enzyme intermediate. The enzyme is then deacylated by a water molecule, and releases the carboxy terminal portion of the peptide. In immunology, papain is known to cleave the Fc (crystallisable) portion of immunoglobulins (antibodies) from the Fab (antigen-binding) portion.

Papain is a relatively heat resistant enzyme, with a temperature optimal range of 60-70°C.^[7]

Papain prefers to cleave at: (hydrophobic)-(Arg or Lys)- cleaves here -(not Val). Hydrophobic is Ala, Val, Leu, Ile, Phe, Trp, or Tyr.^[8]

[edit] Uses

Anthelmintic effect of papain on the worm Heligmosomoides bakeri.

Its utility is in breaking down tough meat fibres and has been used for thousands of years in its native South America. It is sold as a component of powdered meat tenderiser available in most supermarkets. Papain, in the form of such a powdered meat tenderizer, made into a paste with water, is also a home remedy treatment for, bee, and yellow jacket (wasps) stings; mosquito bites; and possibly stingray wounds, breaking down the protein toxins in the venom.^{[citation needed]}

Papain can be used to dissociate cells in the first step of cell culture preparations. A ten-minute treatment of small tissue pieces (less than 1 mm cubed) will allow papain to begin breaking down the extracellular matrix molecules holding the cells together. After ten minutes, the tissue should be treated with a protease inhibitor solution to stop the protease action (if left untreated, papain's activity will lead to complete lysis of the cells). The tissue must then be triturated (passed quickly up and down through a Pasteur pipette) to break up the pieces of tissue into a single cell suspension.

It is also used as an ingredient in various enzymatic debriding preparations, notably Accuzyme. These are used in the care of some chronic wounds to clean up dead tissue.

Papain can also be found as an ingredient in some toothpastes or mints as teeth-whitener. However, its whitening effect in toothpastes and mints is minimal, because the papain is present in low concentrations and it is quickly diluted by saliva. It would take several months of using the whitening product to have noticeably whiter teeth.^{[citation needed]}

It is the main ingredient of Papacarie, a gel used for chemomechanical dental caries removal. Besides the advantage of avoiding the use of rotary cutting tools, it does not interfere in the bond strength of restorative materials to dentin.^[9]

Papain has been known to interfere with urine drug test for cannabinoids.^[10] It is found in some drug detox products.

[edit] Immunoglobulins

The papain-digested antibody: two Fab fragments and an Fc fragment.

An antibody digested by papain yields three fragments: two 50 kDa Fab fragments and one 50kDa Fc fragment. The papain-digested antibody is unable to promote agglutination, precipitation, opsonization, and lysis.

[edit] Production

Papain is usually produced as a crude, dried material by collecting the latex from the fruit of the papaya tree. The latex is collected after scoring the neck of the fruit, where it may either dry on the fruit or drip into a container. This latex is then further dried. It is now classified as a dried, crude material. A purification step is necessary to remove contaminating substances. This purification consists of the solubilization and extraction of the active papain enzyme system through a government-registered process. This purified papain may be supplied as powder or as liquid.

[edit] Action by the FDA to restrict marketing of all opthalmic balanced salt solutions and topical drug products containing papain

See also article: Proteases (medical and related uses)

On September 23, 2008, the FDA warned companies to stop marketing opthalmic balanced salt solutions and topical drug products containing papain by November 4, 2008. The FDA said, "Papain-containing drug products in topical form historically have been marketed without approval...".^[11] According to the FDA's statement on the subject, "These unapproved products have put consumers' health in jeopardy, from reports of permanent vision loss with unapproved balanced salt solutions to a serious drop in blood pressure and increased heart rate from the topical papain products," said Janet Woodcock, M.D., director for the Center for Drug Evaluation and Research. In the same FDA announcement, the FDA states the following:

About Unapproved Topical Papain Products: Topical drug ointments containing papain are used to remove dead or contaminated tissue in acute and chronic lesions, such as diabetic ulcers, pressure ulcers, varicose ulcers, and traumatic infected wounds. Trade names for these products include Accuzyme, Allanfil, Allanzyme, Ethezyme, Gladase, Kovia, Panafil, Pap Urea, and Ziox. Other products are marketed under the names of the active ingredients, for instance, papain-urea ointment.

The FDA is taking action today against these products because it has received reports of serious adverse events in patients using products containing papain. Reports include hypersensitivity (allergic) reactions that lead to hypotension (low blood pressure) and tachycardia (rapid heart rate). In addition, patients who are allergic to latex can also be allergic to papaya, the source of papain. Therefore, patients with latex sensitivity may be at increased risk of suffering an adverse reaction to a topical papain drug product.

FDA urges consumers who are using topical drug products containing papain, and who have questions or concerns, to contact their health care provider about discontinuing treatment with these products. There are a number of FDA-approved topical products that have been found safe and effective as wound healing agents and that do not contain papain.

"Removing unapproved topical drug products containing papain and unapproved ophthalmic balanced salt solutions is yet another step forward for patient safety," said Deborah M. Author, director, Office of Compliance for CDER, FDA.

[edit] Human cysteine proteases from papain family

CTSB; CTSC; CTSF; CTSH; CTSK; CTSL1; CTSL2; CTSL3; CTSO; CTSS; CTSW; CTSZ; TINAG; TINAGL1;

[edit] See also

This section is empty. You can help by adding to it. (April 2012)

[edit] References

[edit] External links

• The MEROPS online database for peptidases and their inhibitors: C01.001
• Papain at the US National Library of Medicine Medical Subject Headings (MeSH)
• drugdigest- info on papin
• Papain Applications, Inhibitors and Substrates
• Data sheet-Papain from BIOZYM