Caricain

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EC no. 3.4.22.30
CAS no. 39307-22-7
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Caricain (EC 3.4.22.30, papaya peptidase A, papaya peptidase II, papaya proteinase, papaya proteinase III, papaya proteinase 3, proteinase omega, papaya proteinase A, chymopapain S, Pp) is an enzyme. [1] [2] [3] [4] [5] [6] This enzyme catalyses the following chemical reaction: Hydrolysis of proteins with broad specificity for peptide bonds, similar to those of papain and chymopapain

Contents

This enzyme is isolated from the papaya plant, Carica papaya .

Name and History

The first description of this enzyme was provided by Schack, [1] who named it papaya peptidase A. The same enzyme has since been given a number of different names, including papaya peptidase II, [4] papaya proteinase III [5] and papaya proteinase. [7] The name caricain was recommended by NC-IUBMB in 1992.

Structural Chemistry

Caricain is synthesized as a preproenzyme. There is evidence at the mRNA level for polymorphism, two very similar clones being isolated, one of which contained a C-terminal extension. [8] The primary structure of the mature form of the enzyme has been determined, [9] and is as predicted from one of the cDNA sequences. The protein is 216 amino acids in length, and is 68% identical in sequence to papain, 65% to chymopapain and 81% to glycyl endopeptidase. The three disulfide bonds are conserved between all the papaya proteinases, and there is no evidence for glycosylation. Caricain is an extremely basic protein, with pI estimated to be 11.7. The A280,1% is reported to be 18.3, giving a molar extinction coefficient of 4.193 104 M21 cm21.

As with some other plant cysteine endopeptidases, caricain exhibits charge heterogeneity. This may be partly due to variation in the oxidization state of the active-site sulfur, as is the case with homologous enzymes from pineapple stem, although genetic polymorphism may also contribute.

The crystal structure of caricain has been solved to a resolution of 1.8 A ̊, and demonstrates main-chain conformation very similar to that of papain. Caricain has four amino acid residues (Ser169-Lys172) not present in papain, but it is papain that is exceptional at this point in the sequence, showing a deletion not seen in other members of the family. The architecture of the active site of caricain is very similar to that of papain.

Preparation

The highly basic character of caricain makes it relatively easy to separate from the other papaya cysteine endopeptidases in cation-exchange chromatography of preparations of commercially available papaya latex. A sodium acetate gradient, pH 5.0, was first used successfully by Robinson [10] and has since been adopted by others. Caricain is found in the latest-eluting protein peak. Due to the charge heterogeneity of caricain the peak may not be symmetrical, but this does not necessarily indicate the presence of contaminants. Covalent chromatography on thiol-Sepharose allows isolation of fully active caricain from the material obtained by cation exchange. [11]

Activity and Specificity

In common with most enzymes in family C1, caricain accepts hydrophobic amino acid residues in both S2 and S3. However, other residues are also accommodated in these subsites, including proline in S2, and lysine in S3. [12] The specificities of three cysteine endopeptidases from papaya latex were found to be very similar. Caricain and chymopapain appeared to prefer an aliphatic to a hydrophobic residue at P2. The similarity in specificity of caricain and chymopapain was demonstrated by the fact that, of 44 peptide bonds in manatee hemoglobin cleaved by caricain, 29 were also cleaved by chymopapain. [12] An earlier study [13] had highlighted the similarity in specificity of caricain, chymopapain and papain. All seven bonds of the oxidized B chain of insulin that were hydrolyzed by caricain were also cleaved by papain, and six were hydrolyzed by chymopapain.

Caricain can be assayed with Bz-ArgkNHPhNO2, kcat/Km being 187 M21 s21 at pH 6.8 and 40˚C. More sensitive substrates may employ a fluorometric leaving group, kcat/Km for the hydrolysis of Z-Phe-ArgkNHMec being 1.06 3 106 M21 s21 (pH 6.8, 40˚C). [5] The enzyme exhibits a broad pH-activity profile, with the optimum near 7.0. About half-maximal activity is still achieved at pH values of about 5.3 and 8.3, and the profile is reported to be governed by at least three ionizing groups. [14] The active-site sulfur requires reduction for catalytic competence, and this is best achieved by the inclusion of low millimolar concentrations of cysteine in assay buffers.

Caricain is inactivated by E-64, making the inhibitor a convenient active-site titrant, and it is inhibited by cystatins, Ki for inhibition by papaya cystatin being 1.5 nM.

Relevant Pharmacokinetics

The structure of procaricain shows a pro-region connected to an active enzyme. [15] Caricain is regarded as a cysteine endopeptidase, that is, it functions through the action of a cysteine residue at its active site and it is capable of hydrolysing peptide bonds that are well within the N-terminus and C-terminus of the substrate.

With proenzymes, the pharmacokinetics would be governed normally by the rate of intramolecular cleavage to produce the active form of the enzyme. The catalytic site is located in a cleft between two lobes and binding of the substrate needs to occur before activity is available. [16] However, as the active form is the one which is present in the processed latex, [17] the rate limiting step in the reaction with proteins will be simply the conversion of the enzyme-substrate complex to product with the regeneration of the enzyme. The hydrolysis of a peptide bond is however, an automatically favourable reaction. [18] Proteolytic enzymes, such as caricain, catalyse the hydrolysis of a peptide bond at rates which depend upon certain chemical groups from amino acids in the neighbourhood of this bond. [19] Hydrolysis is generally confined to peptides made from amino acids of the L-configuration. The rate varies linearly with low substrate concentration (first-order kinetics) and becomes independent at high concentrations of substrate (zero order kinetics).

The kinetics depends upon the rapid formation of an enzyme substrate complex which is then slowly converted to the product in the rate determining step which regenerates the enzyme. Where the concentration of the enzyme is much less than the concentration of the substrate, the rate of reaction is directly proportional to the total enzyme concentration. The hydrolysis of a peptide bond however is an energetically favourable reaction. [18]

Recent experiments with both crude caricain and purified caricain indicated that the reaction which controls detoxification of a wheat gliadin digest at pH7.5 and 37 °C was indeed a 1st order reaction with a rate constant of 1.7 x 10 −4 sec.−1. [20] The rate of reaction was followed by the disappearance of gliadin peptides which were toxic to rat liver lysosomes.

Uses

Gluten is a structural protein naturally found in certain cereal grains, in the medical literature gluten is referred as the combination of prolamin and glutelin proteins naturally occurring in all grains that have been proven capable of triggering celiac disease. Specific immunogenic peptides in gliadin, a class of proteins present in wheat and several other cereals, have the ability to provoke an autoimmune enteropathy caused by an abnormal immune response in genetically susceptible individuals with coeliac disease and other gluten related disorders.

Enzyme therapy for gluten related disorders proposes the use of highly targeted proline and glutamine-specific endoproteases to destroy the immunogenic gluten peptides before these interact with the intestinal lining. In vitro studies [21] [20] demonstrated that caricain offered a high degree of protection against the toxic action of gliadin on rat liver lysosomes and was capable of rapidly digesting the key immuno-reactive gluten epitopes associated with the pathology of celiac disease.

Related Research Articles

<span class="mw-page-title-main">Chymotrypsin</span> Digestive enzyme

Chymotrypsin (EC 3.4.21.1, chymotrypsins A and B, alpha-chymar ophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase, alpha-chymar, alpha-chymotrypsin A, alpha-chymotrypsin) is a digestive enzyme component of pancreatic juice acting in the duodenum, where it performs proteolysis, the breakdown of proteins and polypeptides. Chymotrypsin preferentially cleaves peptide amide bonds where the side chain of the amino acid N-terminal to the scissile amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine). These amino acids contain an aromatic ring in their side chain that fits into a hydrophobic pocket (the S1 position) of the enzyme. It is activated in the presence of trypsin. The hydrophobic and shape complementarity between the peptide substrate P1 side chain and the enzyme S1 binding cavity accounts for the substrate specificity of this enzyme. Chymotrypsin also hydrolyzes other amide bonds in peptides at slower rates, particularly those containing leucine at the P1 position.

<span class="mw-page-title-main">Proteolysis</span> Breakdown of proteins into smaller polypeptides or amino acids

Proteolysis is the breakdown of proteins into smaller polypeptides or amino acids. Uncatalysed, the hydrolysis of peptide bonds is extremely slow, taking hundreds of years. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion.

<span class="mw-page-title-main">Protease</span> Enzyme that cleaves other proteins into smaller peptides

A protease is an enzyme that catalyzes proteolysis, breaking down proteins into smaller polypeptides or single amino acids, and spurring the formation of new protein products. They do this by cleaving the peptide bonds within proteins by hydrolysis, a reaction where water breaks bonds. Proteases are involved in numerous biological pathways, including digestion of ingested proteins, protein catabolism, and cell signaling.

In biology and biochemistry, protease inhibitors, or antiproteases, are molecules that inhibit the function of proteases. Many naturally occurring protease inhibitors are proteins.

<span class="mw-page-title-main">Papain</span> Widely used enzyme extracted from papayas

Papain, also known as papaya proteinase I, is a cysteine protease enzyme present in papaya and mountain papaya. It is the namesake member of the papain-like protease family.

<span class="mw-page-title-main">Catalytic triad</span> Set of three coordinated amino acids

A catalytic triad is a set of three coordinated amino acids that can be found in the active site of some enzymes. Catalytic triads are most commonly found in hydrolase and transferase enzymes. An acid-base-nucleophile triad is a common motif for generating a nucleophilic residue for covalent catalysis. The residues form a charge-relay network to polarise and activate the nucleophile, which attacks the substrate, forming a covalent intermediate which is then hydrolysed to release the product and regenerate free enzyme. The nucleophile is most commonly a serine or cysteine amino acid, but occasionally threonine or even selenocysteine. The 3D structure of the enzyme brings together the triad residues in a precise orientation, even though they may be far apart in the sequence.

<span class="mw-page-title-main">Cysteine protease</span> Class of enzymes

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

<span class="mw-page-title-main">Aspartic protease</span>

Aspartic proteases are a catalytic type of protease enzymes that use an activated water molecule bound to one or more aspartate residues for catalysis of their peptide substrates. In general, they have two highly conserved aspartates in the active site and are optimally active at acidic pH. Nearly all known aspartyl proteases are inhibited by pepstatin.

<span class="mw-page-title-main">Ficain</span>

Ficain also known as ficin, debricin, or higueroxyl delabarre is a proteolytic enzyme extracted from the latex sap from the stems, leaves, and unripe fruit of the American wild fig tree Ficus insipida.

<span class="mw-page-title-main">Actinidain</span> Class of enzymes

Actinidain is a type of cysteine protease enzyme found in fruits including kiwifruit, pineapple, mango, banana, figs, and papaya. This enzyme is part of the peptidase C1 family of papain-like proteases.

<span class="mw-page-title-main">Proteinase K</span> Broad-spectrum serine protease

In molecular biology, Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album. Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons.

<span class="mw-page-title-main">Chymopapain</span> Enzyme

Chymopapain is a proteolytic enzyme isolated from the latex of papaya. It is a cysteine protease which belongs to the papain-like protease (PLCP) group. Because of its proteolytic activity, it is the main molecule in the process of chemonucleolysis, used in some procedures like the treatment of herniated lower lumbar discs in the spine by a nonsurgical method.

Cathepsin X is an enzyme. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Oligopeptidase</span> Enzymes that cleaves peptides but not proteins

An Oligopeptidase is an enzyme that cleaves peptides but not proteins. This property is due to its structure: the active site of this enzyme is located at the end of a narrow cavity which can only be reached by peptides.

Glycyl endopeptidase is an enzyme. This enzyme catalyses the following chemical reaction

<span class="mw-page-title-main">Zingibain</span>

Zingibain, zingipain, or ginger protease is a cysteine protease enzyme found in ginger rhizomes. It catalyses the preferential cleavage of peptides with a proline residue at the P2 position. It has two distinct forms, ginger protease I (GP-I) and ginger protease II (GP-II).

<span class="mw-page-title-main">Scytalidopepsin B</span>

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Asparagine peptide lyase are one of the seven groups in which proteases, also termed proteolytic enzymes, peptidases, or proteinases, are classified according to their catalytic residue. The catalytic mechanism of the asparagine peptide lyases involves an asparagine residue acting as nucleophile to perform a nucleophilic elimination reaction, rather than hydrolysis, to catalyse the breaking of a peptide bond.

<span class="mw-page-title-main">Thimet oligopeptidase</span>

Thimet oligopeptidases, also known as TOPs, are a type of M3 metallopeptidases. These enzymes can be found in animals and plants, showing distinctive functions. In animals and humans, they are involved in the degradation of peptides, such as bradykinin, neurotensin, angiotensin I, and Aβ peptide, helping to regulate physiological processes. In plants, their role is related to the degradation of targeting peptides and the immune response to pathogens through Salicylic Acid (SA)-dependent stress signaling. In Arabidopsis thaliana—recognized as a model plant for scientific studies—two thimet oligopeptidases, known as TOP1 and TOP2, have been identified as targets for salicylic acid binding in the plant. These TOP enzymes are key components to understand the SA-mediated signaling where interactions exist with different components and most of the pathways are unknown.

<span class="mw-page-title-main">Asparagine endopeptidase</span> Class of enzymes

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References

  1. 1 2 Schack P (1967). "Fractionation of proteolytic enzymes of dried papaya latex. Isolation and preliminary characterization of a new proteolytic enzyme". Comptes Rendus des Travaux du Laboratoire Carlsberg. 36 (4): 67–83. PMID   6043136.
  2. ↑ Robinson GW (August 1975). "Isolation and characterization of papaya peptidase A from commercial chymopapain". Biochemistry. 14 (16): 3695–700. doi:10.1021/bi00687a028. PMID   240390.
  3. ↑ Polgár L (July 1984). "Problems of classification of papaya latex proteinases". The Biochemical Journal. 221 (2): 555–6. doi:10.1042/bj2210555. PMC   1144075 . PMID   6383350.
  4. 1 2 Brocklehurst K, Salih E, McKee R, Smith H (June 1985). "Fresh non-fruit latex of Carica papaya contains papain, multiple forms of chymopapain A and papaya proteinase omega". The Biochemical Journal. 228 (2): 525–7. doi:10.1042/bj2280525. PMC   1145013 . PMID   4015629.
  5. 1 2 3 Zucker S, Buttle DJ, Nicklin MJ, Barrett AJ (April 1985). "The proteolytic activities of chymopapain, papain, and papaya proteinase III". Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 828 (2): 196–204. doi:10.1016/0167-4838(85)90057-3. PMID   3919769.
  6. ↑ Dubois T, Kleinschmidt T, Schnek AG, Looze Y, Braunitzer G (August 1988). "The thiol proteinases from the latex of Carica papaya L. II. The primary structure of proteinase omega". Biological Chemistry Hoppe-Seyler. 369 (8): 741–54. doi:10.1515/bchm3.1988.369.2.741. PMID   3063283.
  7. ↑ Brocklehurst, K., Salih, E., McKee, R., Smith, H. (1985). Fresh non-fruit latex of Carica papaya contains papain, multiple forms of chymopapain A and papaya proteinase S. Biochem. J. 228, 525–527.
  8. ↑ Revell, D.F., Cummings, N.J., Baker, K.C., Collins, M.E., Taylor, M.A.J., Sumner, I.G., Pickersgill, R.W., Connerton, I.F., Goodenough, P.W. (1993). Nucleotide sequence and expression in Escherichia coli of cDNAs encoding papaya proteinase Ω from Carica papaya. Gene 127, 221–225.
  9. ↑ Dubois, T., Kleinschmidt, T., Schnek, A.G., Looze, Y., Braunitzer, G. (1988). The thiol proteinases Ω from the latex of Carica papaya L. II. The primary structure of proteinase. Biol. Chem. Hoppe-Seyler 369, 741–754.
  10. ↑ Robinson, G.W. (1975). Isolation and characterization of papaya pepti- dase A from commercial chymopapain. Biochemistry 14, 3695–3700.
  11. ↑ Dubois, T., Jacquet, A., Schnek, A.G., Looze, Y. (1988). The thiol proteinases from the latex of Carica papaya L. I. Fractionation, purification and preliminary characterization. Biol. Chem. Hoppe- Seyler 369, 733–740.
  12. 1 2 Jacquet, A., Kleinschmidt, T., Dubois, T., Schnek, A.G., Looze, Y., Braunitzer, G. (1989). The thiol proteinases from the latex of Carica papaya L. IV. Proteolytic specificities of chymopa- pain and papaya Ω proteinase determined by digestion of α-globin chains. Biol. Chem. Hoppe-Seyler 370, 819–829.
  13. ↑ Johansen, J.T., Ottesen, M. (1968). The proteolytic degradation of the B-chain of oxidized insulin by papain, chymopapain and papaya peptidase. Comptes Rendus Lab. Carlsberg 36, 265–283.
  14. ↑ Taylor, M.A.J., Baker, K.C., Connerton, I.F., Cummings, N.J., Harris, G.W., Henderson, I.M.J., Jones, S.T., Pickersgill, R.W., Sumner, I.G., Warwicker, J., Goodenough, P.W. (1994). An unequivocal example of cysteine proteinase activity affected by multiple electrostatic interactions. Protein Eng. 7, 1267–1276.
  15. ↑ Groves, R.H., Hosking, J.R., Batianoff, G.N., Cooke, D.A., Cowie, I.D., Johnson, R.W., Keighery, G.J., Lepschi, B.J., Mitchell, A.A., Moerkerk, M., Randall, R.P., Rozefelds, A.C., Walsh, N.G., Waterhouse, B.M. (2003). Weed categories for natural and agricultural ecosystem management. Bureau of Rural Sciences, Australian Government Department of Agriculture, Fisheries and Forestry, Canberra.
  16. ↑ Pickersgill, Barbara. "Cytogenetics and evolution of Capsicum L." Chromosome engineering in plants: genetics, breeding, evolution, part B. Elsevier, Amsterdam (1991): 139-160.
  17. ↑ Azarkan, M., El Moussaoui, A., Van Wuytswinkel, D., Dehon, G. & Looze, Y. (2003). Fractionation and purification of the enzymes stored in the latex of Carica papaya. J Chromatog.B Vol. 790. pp. 229-238
  18. 1 2 Snider, Mark J.; Wolfenden, Richard (2001-08-30). "Site-Bound Water and the Shortcomings of a Less than Perfect Transition State Analogue". Biochemistry. 40 (38): 11364–11371. doi:10.1021/bi011189+. ISSN   0006-2960. PMID   11560484.
  19. ↑ Laidler KJ. Physical chemistry with biological applications. Menlo Park, CA: Benjamin Cummings, 1978; p. 427–451.
  20. 1 2 Cornell, Hugh J.; Stelmasiak, Teodor (2011). "Caricain: A basis for enzyme therapy for coeliac disease". South African Journal of Science. 107 (9/10). doi: 10.4102/sajs.v107i9/10.529 .
  21. ↑ Cornell, H. J.; Doherty, W.; Stelmasiak, T. (2009-01-21). "Papaya latex enzymes capable of detoxification of gliadin". Amino Acids. 38 (1): 155–165. doi:10.1007/s00726-008-0223-6. ISSN   0939-4451.
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