Papain: Difference between revisions
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[[Image:Papain_cartoon.png|200px|left|thumb|Cartoon image of papain.]] | [[Image:Papain_cartoon.png|200px|left|thumb|Cartoon image of papain.]] | ||
'''Papain''' is a 23.4 kDa, 212 residue cysteine protease, also known as '''papaya proteinase I''', from the peptidase C1 family (E.C. 3.4.22.2).<ref name="UniProt">http://www.uniprot.org/uniprot/P00784</ref><ref name="9PAP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP] 9PAP PDB</ref> It is the natural product of the [http://en.wikipedia.org/wiki/Carica_papaya Papaya](''Carica papaya'')<ref name="Sigma Aldrich">http://http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref>, and may be extracted from the plant's latex, leaves and roots.<ref name="worthington">http://www.worthington-biochem.com/pap/default.html</ref> Papain displays a broad range of functions, acting as an endopeptidase, amidase, and esterase,<ref name="Worthington">http://www.worthington-biochem.com/pap/default.html</ref> with its optimal activity values for pH lying between 6.0 and 7.0, and its optimal temperature for activity is 65 °C. Its pI values are 8.75 and 9.55, and it is best visualized at a wavelength of 278 nm.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> | '''Papain''' is a 23.4 kDa, 212 residue cysteine protease, also known as '''papaya proteinase I''', from the peptidase C1 family (E.C. 3.4.22.2).<ref name="UniProt">http://www.uniprot.org/uniprot/P00784</ref><ref name="9PAP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP] 9PAP PDB</ref> It is the natural product of the [http://en.wikipedia.org/wiki/Carica_papaya Papaya](''Carica papaya'')<ref name="Sigma Aldrich">http://http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref>, and may be extracted from the plant's latex, leaves and roots.<ref name="worthington">http://www.worthington-biochem.com/pap/default.html</ref> Papain displays a broad range of functions, acting as an endopeptidase, amidase, and esterase,<ref name="Worthington">http://www.worthington-biochem.com/pap/default.html</ref> with its optimal activity values for pH lying between 6.0 and 7.0, and its optimal temperature for activity is 65 °C. Its pI values are 8.75 and 9.55, and it is best visualized at a wavelength of 278 nm.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> | ||
Papain's enzymatic use was first discovered in 1873 by G.C. Roy who published his results in the Calcutta Medical Journal in the article, "The Solvent Action of Papaya Juice on Nitrogenous Articles of Food." In 1879, papain was named officially by Wurtz and Bouchut, who managed to partially purify the product from the sap of papaya. It wasn't until the mid-twentieth century that the complete purification and isolation of papain was achieved. In 1968, Drenth et al. determined the structure of papain by x-ray crystallography, making it the second enzyme whose structure was successfully determined by x-ray crystallography. Additionally, papain was the first cysteine protease to have its structure identified.<ref name="Worthington" /> In 1984, Kamphuis et al. determined the geometry of the active site, and the three-dimensional structure was visualized to a 1.65 Angstrom solution.<ref name="Structure">PMID:6502713</ref> Today, studies continue on the stability of papain, involving changes in environmental conditions as well as testing of inhibitors such as phenylmethanesulfonylfluoride (PMSF), TLCK, TPCK, aplh2-macroglobulin, heavy metals, AEBSF, antipain, cystatin, E-64, leupeptin, sulfhydryl binding agents, carbonyl reagents, and alkylating agents.<ref name="Worthington" /> | Papain's enzymatic use was first discovered in 1873 by G.C. Roy who published his results in the Calcutta Medical Journal in the article, "The Solvent Action of Papaya Juice on Nitrogenous Articles of Food." In 1879, papain was named officially by Wurtz and Bouchut, who managed to partially purify the product from the sap of papaya. It wasn't until the mid-twentieth century that the complete purification and isolation of papain was achieved. In 1968, Drenth et al. determined the structure of papain by x-ray crystallography, making it the second enzyme whose structure was successfully determined by x-ray crystallography. Additionally, papain was the first cysteine protease to have its structure identified.<ref name="Worthington" /> In 1984, Kamphuis et al. determined the geometry of the active site, and the three-dimensional structure was visualized to a 1.65 Angstrom solution.<ref name="Structure">PMID:6502713</ref> Today, studies continue on the stability of papain, involving changes in environmental conditions as well as testing of inhibitors such as phenylmethanesulfonylfluoride (PMSF), TLCK, TPCK, aplh2-macroglobulin, heavy metals, AEBSF, antipain, cystatin, E-64, leupeptin, sulfhydryl binding agents, carbonyl reagents, and alkylating agents.<ref name="Worthington" /> | ||
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==Structure==<StructureSection load='9pap' size='500' side='right' caption='Structure of Papain (PDB entry [[9pap]])' scene=''>Anything in this section will appear adjacent to the 3D structure and will be scrollable. | ==Structure==<StructureSection load='9pap' size='500' side='right' caption='Structure of Papain (PDB entry [[9pap]])' scene=''>Anything in this section will appear adjacent to the 3D structure and will be scrollable. | ||
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In order to investigate binding of protein substrates to papain, Schröder et. al. crystallized the enzyme with the broad-spectrum competitive protease inhibitor leupeptin, shown in blue in the ribbon diagram. It has the structure Ac-Leu-Leu-Arginal, where Ac is an acetyl group attached to the nitrogen of the first leucine. The inhibitor functions by binding to the enzyme's active site, where the catalytic nucleophile (cysteine in papain) attacks the arginal aldehyde. This forms a tight-binding transition state from which the normal catalytic mechanism cannot proceed, due to this carbonyl having no potential leaving groups bonded to it. Analysis of the resulting structure revealed that the <scene name='9pap/Papain_sam_1popactivesite/1'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='9pap/1pop_sam_leupeptin_hydrophobic/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make <scene name='9pap/1pop_sam_leupeptin_hbonds/1'>hydrogen bonds</scene> with some of the leupeptin atoms. These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both Gln-19 and the amide nitrogen of the catalytic Cys-25 with the arginal carbanion, forming the catalytically important oxyanion hole. In addition, Gly-66 interacts with the second leucine in leupeptin while Asp-158 interacts with a hydrogen on the arginal. These interaction further stabilize and orient the substrate in the binding pocket<ref name="Schroder">[http://www.sciencedirect.com/science/article/pii/001457939381128M] Schröder, E., C. Phillips, E. Garman, K. Harlos, C. Crawford. 1997. X-ray crystallographic structure of a papain-leupeptin complex. FEBS Letters 315: 38-42</ref>. | In order to investigate binding of protein substrates to papain, Schröder et. al. crystallized the enzyme with the broad-spectrum competitive protease inhibitor leupeptin, shown in blue in the ribbon diagram. It has the structure Ac-Leu-Leu-Arginal, where Ac is an acetyl group attached to the nitrogen of the first leucine. The inhibitor functions by binding to the enzyme's active site, where the catalytic nucleophile (cysteine in papain) attacks the arginal aldehyde. This forms a tight-binding transition state from which the normal catalytic mechanism cannot proceed, due to this carbonyl having no potential leaving groups bonded to it. Analysis of the resulting structure revealed that the <scene name='9pap/Papain_sam_1popactivesite/1'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='9pap/1pop_sam_leupeptin_hydrophobic/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make <scene name='9pap/1pop_sam_leupeptin_hbonds/1'>hydrogen bonds</scene> with some of the leupeptin atoms. These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both Gln-19 and the amide nitrogen of the catalytic Cys-25 with the arginal carbanion, forming the catalytically important oxyanion hole. In addition, Gly-66 interacts with the second leucine in leupeptin while Asp-158 interacts with a hydrogen on the arginal. These interaction further stabilize and orient the substrate in the binding pocket<ref name="Schroder">[http://www.sciencedirect.com/science/article/pii/001457939381128M] Schröder, E., C. Phillips, E. Garman, K. Harlos, C. Crawford. 1997. X-ray crystallographic structure of a papain-leupeptin complex. FEBS Letters 315: 38-42</ref>. | ||
===Medicinal Uses=== | |||
Papain has been used for a plethora of medicinal purposes including treating inflammation, shingles, diarrhea, psoriasis, parasites, and many others.<ref>http://www.webmd.com/vitamins-supplements/ingredientmono-69-PAPAIN.aspx?activeIngredientId=69&activeIngredientName=PAPAIN</ref> One major use is the treatment of cutaneous ulcers including diabetic ulcers and pressure ulcers.<ref>http://www.pbm.va.gov/Clinical%20Guidance/Drug%20Monographs/Papain%20Urea.pdf</ref> Pressures ulcers plague many bed bound individuals and are a major source of pain and discomfort. Two papain based topical drugs are Accuzyme and Panafil, which can be used to treat wounds like cutaneous ulcers.<ref>http://www.pbm.va.gov/Clinical%20Guidance/Drug%20Monographs/Papain%20Urea.pdf</ref> | |||
[[Image:papayas.jpg|150px|right|thumb|Papaya<ref>http://dailyfitnessmagz.com/2011/03/papayas-nutrition-facts/</ref>]] | |||
A recent New York Times article featured papain and other digestive enzymes.<ref>http://www.nytimes.com/2012/02/23/fashion/enzymes-once-sidelined-try-to-grab-the-spotlight.html</ref> With the number of individuals suffering from irritable bowel syndrome and other gastrointestinal issues, many people are turning toward natural digestive aid supplements like papain. The author even talks about the use of papain along with a pineapple enzyme, bromelain, in cosmetic facial masks. Dr. Adam R. Kolker (a plastic surgeon) is quoted in the article saying that "For skin that is sensitive, enzymes are wonderful." He bases these claims off the idea that proteases like papain help to break peptide bonds holding dead skin cells to the live skin cells.<ref>http://www.nytimes.com/2012/02/23/fashion/enzymes-once-sidelined-try-to-grab-the-spotlight.html</ref> | |||
[[Image:2fab_fc.png|150px|right|thumb|Diagram showing cleavage products of an antibody digested by papain.]] | |||
Papain digests most proteins, often more extensively than pancreatic proteases. It has a very broad specificity and is known to cleave peptide bonds of basic amino acids and leucine and glycine residues, but prefers amino acids with large hydrophobic side chains. This non-specific nature of papain's hydrolase activity has led to its use in many and varied commercial products. It is often used as a meat tenderizer because it can hydrolyze the peptide bonds of collagen, elastin, and actomyosin. It is also used in contact lens solution to remove protein deposits on the lenses and marketed as a digestive supplement. Another place papain has found use is clinical medicine, where it is used to treat pain, swelling, and fluid retention following trauma and surgery. <ref> http://www.webmd.com/vitamins-supplements/ingredientmono-69-PAPAIN.aspx?activeIngredientId=69&activeIngredientName=PAPAIN </ref> Finally, papain has several common uses in general biomedical research, including a gentle cell isolation agent, production of glycopeptides from purified proteoglycans, and solubilization of integral membrane proteins. It is also notable for its ability to specifically cleave IgG and IgM antibodies above and below the disulfide bonds that join the heavy chains and that is found between the light chain and heavy chain. This generates two monovalent Fab segments, that each have a single antibody binding sites, and an intact Fc fragment, as shown in the image to the right:<ref name="Worthington" /> | |||
==Reference== | ==Reference== | ||
