Caricain (, ''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 An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

. This enzyme catalyses the following

chemical reaction A chemical reaction is a process that leads to the chemistry, chemical transformation of one set of chemical substances to another. When chemical reactions occur, the atoms are rearranged and the reaction is accompanied by an Gibbs free energy, ...

Hydrolysis Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

of proteins with broad specificity for

peptide bond In organic chemistry, a peptide bond is an amide type of covalent chemical bond linking two consecutive alpha-amino acids from C1 (carbon number one) of one alpha-amino acid and N2 (nitrogen number two) of another, along a peptide or protein cha ...

s, similar to those of

papain Papain, also known as papaya proteinase I, is a cysteine protease () enzyme present in papaya (''Carica papaya'') and mountain papaya (''Vasconcellea cundinamarcensis''). It is the namesake member of the papain-like protease family. It has wi ...

and

chymopapain Chymopapain (, ''chymopapain A'', ''chymopapain B'', ''chymopapain S'', brand name Chymodiactin) is a proteolytic enzyme isolated from the latex of papaya (''Carica papaya''). It is a cysteine protease which belongs to the papain-like protease (PL ...

This enzyme is isolated from the

papaya The papaya (, ), papaw, () or pawpaw () is the plant species ''Carica papaya'', one of the 21 accepted species in the genus '' Carica'' of the family Caricaceae, and also the name of its fruit. It was first domesticated in Mesoamerica, within ...

plant, ''

Carica papaya The papaya (, ), papaw, () or pawpaw () is the plant species ''Carica papaya'', one of the 21 accepted species in the genus ''Carica'' of the family Caricaceae, and also the name of its fruit. It was first domesticated in Mesoamerica, within ...

''.

Name and History

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

Structural Chemistry

Caricain is synthesized as a

preproenzyme A preproenzyme is an enzyme with two additional characteristics: "pre" refers to a signal sequence (signal peptide) which directs the enzyme to a specific organelle or subcellular localization; "pro" indicates that the enzyme is present in an inacti ...

. There is evidence at the mRNA level for polymorphism, two very similar clones being isolated, one of which contained a

C-terminal The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

extension. The primary structure of the mature form of the enzyme has been determined, and is as predicted from one of the

cDNA In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...

sequences. The protein is 216 amino acids in length, and is 68% identical in sequence to

, 65% to

and 81% to

glycyl endopeptidase Glycyl endopeptidase (, ''papaya peptidase B'', ''papaya proteinase IV'', ''glycine-specific proteinase'', '' chymopapain'', ''Papaya proteinase 4'', ''PPIV'', ''chymopapain M'') is an enzyme. This enzyme catalyses the following chemical reaction ...

. The three

disulfide bonds In chemistry, a disulfide (or disulphide in British English) is a compound containing a functional group or the anion. The linkage is also called an SS-bond or sometimes a disulfide bridge and usually derived from two thiol groups. In in ...

are conserved between all the papaya proteinases, and there is no evidence for

glycosylation Glycosylation is the reaction in which a carbohydrate (or ' glycan'), i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor) in order to form a glycoconjugate. In biology (but not ...

. 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 A gene is said to be polymorphic if more than one allele occupies that gene's locus within a population. In addition to having more than one allele at a specific locus, each allele must also occur in the population at a rate of at least 1% to ge ...

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

, 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 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 Sepharose is a tradename for a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed. Its brand name is a portmanteau derived from Separation-Pharmacia-Agarose. A common application for the material is in chro ...

allows isolation of fully active caricain from the material obtained by cation exchange.

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.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. The specificities of three cysteine endopeptidases from papaya latex were found to be very similar. Caricain and

appeared to prefer an

aliphatic In organic chemistry, hydrocarbons ( compounds composed solely of carbon and hydrogen) are divided into two classes: aromatic compounds and aliphatic compounds (; G. ''aleiphar'', fat, oil). Aliphatic compounds can be saturated (in which all ...

to a

hydrophobic In chemistry, hydrophobicity is the chemical property of a molecule (called a hydrophobe) that is seemingly repelled from a mass of water. In contrast, hydrophiles are attracted to water. Hydrophobic molecules tend to be nonpolar and, thu ...

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. An earlier study 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). 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. 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

cystatin The cystatins are a family of cysteine protease inhibitors which share a sequence homology and a common tertiary structure of an alpha helix lying on top of an anti-parallel beta sheet. The family is subdivided as described below. Cystatins sho ...

s, 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. Caricain is regarded as a

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

endopeptidase Endopeptidase or endoproteinase are proteolytic peptidases that break peptide bonds of nonterminal amino acids (i.e. within the molecule), in contrast to exopeptidases, which break peptide bonds from end-pieces of terminal amino acids. For this r ...

, 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 The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

and

C-terminus The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein Proteins are large biomolecules and macromolecules that comp ...

of the

substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (aquatic environment), the earthy material that exi ...

. With proenzymes, the

pharmacokinetics Pharmacokinetics (from Ancient Greek ''pharmakon'' "drug" and ''kinetikos'' "moving, putting in motion"; see chemical kinetics), sometimes abbreviated as PK, is a branch of pharmacology dedicated to describing how the body affects a specific su ...

would be governed normally by the rate of intramolecular cleavage to produce the active form of the enzyme. The

catalytic site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding si ...

is located in a cleft between two lobes and binding of the substrate needs to occur before activity is available. However, as the active form is the one which is present in the processed latex, 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 Hydrolysis (; ) is any chemical reaction in which a molecule of water breaks one or more chemical bonds. The term is used broadly for substitution reaction, substitution, elimination reaction, elimination, and solvation reactions in which water ...

of a

is however, an automatically favourable reaction. 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. 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. Recent experiments with both crude caricain and purified caricain indicated that the reaction which controls detoxification of a wheat

gliadin Gliadin (a type of prolamin) is a class of proteins present in wheat and several other cereals within the grass genus ''Triticum''. Gliadins, which are a component of gluten, are essential for giving bread the ability to rise properly during ba ...

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. The rate of reaction was followed by the disappearance of gliadin

peptide Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty am ...

s which were toxic to rat liver lysosomes.

Uses

Gluten related disorders

Gluten Gluten is a structural protein naturally found in certain Cereal, cereal grains. The term ''gluten'' usually refers to the elastic network of a wheat grain's proteins, gliadin and glutenin primarily, that forms readily with the addition of water ...

is a structural

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

naturally found in certain cereal grains, in the medical literature gluten is referred as the combination of

prolamin Prolamins are a group of plant storage proteins having a high proline amino acid content. They are found in plants, mainly in the seeds of cereal grains such as wheat ( gliadin), barley ( hordein), rye ( secalin), corn ( zein), sorghum ( kafiri ...

and

glutelin Glutelins are a class of prolamin proteins found in the endosperm of certain seeds of the grass family. They constitute a major component of the protein composite collectively referred to as gluten. Glutenin is the most common glutelin, as it is ...

proteins naturally occurring in all grains that have been proven capable of triggering

celiac disease Coeliac disease (British English) or celiac disease (American English) is a long-term autoimmune disorder, primarily affecting the small intestine. Patients develop intolerance to gluten, which is present in foods such as wheat, rye, spel ...

. Specific immunogenic peptides in

, a class of

proteins Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, re ...

present in

wheat Wheat is a group of wild and crop domestication, domesticated Poaceae, grasses of the genus ''Triticum'' (). They are Agriculture, cultivated for their cereal grains, which are staple foods around the world. Well-known Taxonomy of wheat, whe ...

and several other

cereal A cereal is a grass cultivated for its edible grain. Cereals are the world's largest crops, and are therefore staple foods. They include rice, wheat, rye, oats, barley, millet, and maize ( Corn). Edible grains from other plant families, ...

s, have the ability to provoke an autoimmune enteropathy caused by an abnormal immune response in genetically susceptible individuals with

coeliac disease Coeliac disease (British English) or celiac disease (American English) is a long-term autoimmune disorder, primarily affecting the small intestine. Patients develop intolerance to gluten, which is present in foods such as wheat, rye, spelt ...

and other gluten related disorders. Enzyme therapy for gluten related disorders proposes the use of highly targeted

proline Proline (symbol Pro or P) is an organic acid classed as a proteinogenic amino acid (used in the biosynthesis of proteins), although it does not contain the amino group but is rather a secondary amine. The secondary amine nitrogen is in the p ...

and

glutamine Glutamine (symbol Gln or Q) is an α-amino acid that is used in the biosynthesis of proteins. Its side chain is similar to that of glutamic acid, except the carboxylic acid group is replaced by an amide. It is classified as a charge-neutral ...

-specific endoproteases to destroy the immunogenic gluten peptides before these interact with the intestinal lining. In vitro studies 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.

References

External links

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