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== Papain == | == Papain == | ||
<Structure load='9pap' size='350' frame='true' align='right' caption='Papain' scene='Sandbox_34/Entire_protein_with_ligandscys/2' /> | |||
=== Introduction === | |||
'''Papain''' is a 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> It functions 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> While only consisting of a single peptide chain, papain has <scene name='Sandbox_34/Subunitsrandl/1'>two domains</scene> that form a cleft in which the <scene name='Sandbox_34/2subuwithactivesite/4'>active site</scene> lies.<ref name="PDBSum">http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=9pap&template=clefts.html&r=speedfill</ref> Naturally found in the latex of the papaya fruit, one of the most common uses of papain is as a meat tenderizer because of its ability to hydrolyze esters and amides.<ref>IUBMB Enzyme Nomenclature: www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/22/2.html</ref> Another common use is as a digestive aid. Papaya is commonly referenced as a preferred fruit for those suffering from gastroesophageal reflux disease due to its ability to help the the stomach with digestion of complex proteins. | |||
'''Papain''' is a 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> It functions 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> While only consisting of a single peptide chain, papain has <scene name='Sandbox_34/Subunitsrandl/1'>two domains</scene> that form a cleft in which the <scene name='Sandbox_34/2subuwithactivesite/4'>active site</scene> lies.<ref name="PDBSum">http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=9pap&template=clefts.html&r=speedfill</ref> Naturally found in the latex of the papaya fruit, one of the most common uses of papain is as a meat tenderizer because of its ability to hydrolyze esters and amides.<ref>IUBMB Enzyme Nomenclature: www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/22/2.html</ref> Another common use is as a digestive aid. Papaya is commonly referenced as a preferred fruit for those suffering from gastroesophageal reflux disease due to its ability to help the the stomach with digestion of complex proteins. | |||
=== History === | === History === | ||
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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" /> | ||
== Structure == | == Structure == | ||
<Structure load='9pap' size='375' frame='true' align='left' caption='Papain and Structure' scene='Sandbox_34/Subunitsrandl/3'/> | |||
Papain is a relatively simple enzyme. It consists of only one chain of 212 residues with three disulfide bonds, illustrated in yellow. The single chain is separated into <scene name='Sandbox_34/Subunitsrandl/1'>two domains</scene>: R is shown in purple, and L in gray. A cleft is formed in which the <scene name='Sandbox_34/2subuwithactivesite/4'>active site</scene>, consisting of cysteine-25, histidine-159, and asparagine-175, resides.<ref name="PDBSum" /> Many hydrogen bonds exist throughout the molecule, holding it in its 3D conformation. These stabilizing bonds, represented in white, are present both in the <scene name='Sandbox_34/Pap_with_h-bonding_btwnbckbne/2'>backbone</scene>, and between <scene name='Sandbox_34/Pap_with_h-bonding_btwnsdchns/3'>residues</scene>. | Papain is a relatively simple enzyme. It consists of only one chain of 212 residues with three disulfide bonds, illustrated in yellow. The single chain is separated into <scene name='Sandbox_34/Subunitsrandl/1'>two domains</scene>: R is shown in purple, and L in gray. A cleft is formed in which the <scene name='Sandbox_34/2subuwithactivesite/4'>active site</scene>, consisting of cysteine-25, histidine-159, and asparagine-175, resides.<ref name="PDBSum" /> Many hydrogen bonds exist throughout the molecule, holding it in its 3D conformation. These stabilizing bonds, represented in white, are present both in the <scene name='Sandbox_34/Pap_with_h-bonding_btwnbckbne/2'>backbone</scene>, and between <scene name='Sandbox_34/Pap_with_h-bonding_btwnsdchns/3'>residues</scene>. | ||
<scene name='Sandbox_34/Salt_bridges/5'>Salt bridges</scene> also strongly contribute to the stability of the protein structure. In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue A modified cysteine residue with a sulfhydryl group, <scene name='Sandbox_34/9pap_sulfhydryl_group/1'>cysteine sulfonic acid</scene>, is necessary for the activity of the enzyme<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In 9PAP, the primary representation of papain used in this article, the sulfhydryl group has been oxidized. Papain contains many <scene name='Sandbox_34/Hydrophobicpolar/1'>hydrophobic and polar regions</scene>. The <scene name='Sandbox_34/Hydrophobic_residues/1'>hydrophobic residues</scene> are illustrated in gray, and the <scene name='Sandbox_34/Polar_residues/1'>polar residues</scene> are illustrated in magenta. It is easy to see that the hydrophobic and polar residues segregate themselves such that hydrophobic residues are buried within turns or the interior of the molecule, and that polar regions are towards the exterior of the molecule. | <scene name='Sandbox_34/Salt_bridges/5'>Salt bridges</scene> also strongly contribute to the stability of the protein structure. In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue A modified cysteine residue with a sulfhydryl group, <scene name='Sandbox_34/9pap_sulfhydryl_group/1'>cysteine sulfonic acid</scene>, is necessary for the activity of the enzyme<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In 9PAP, the primary representation of papain used in this article, the sulfhydryl group has been oxidized. Papain contains many <scene name='Sandbox_34/Hydrophobicpolar/1'>hydrophobic and polar regions</scene>. The <scene name='Sandbox_34/Hydrophobic_residues/1'>hydrophobic residues</scene> are illustrated in gray, and the <scene name='Sandbox_34/Polar_residues/1'>polar residues</scene> are illustrated in magenta. It is easy to see that the hydrophobic and polar residues segregate themselves such that hydrophobic residues are buried within turns or the interior of the molecule, and that polar regions are towards the exterior of the molecule.In a paper entitled, ''The Structure of Papain Refined at 1.65 A Resoltion'', Kamphuis et al. discovered interesting information on <scene name='Sandbox_34/All_bonding_shenanigans/1'>direct protein-protein contacts</scene> between molecules of papain in solution. These contacts, communicated in Table 7 of their paper, consist of nine hydrogen-bond connections and three ionic interactions. The strongest salt bridge exists between <scene name='Sandbox_34/Arg191asp140intraxn/2'>asparagine-140 and arginine-191</scene>.<ref name="Structure" /> | ||
In a paper entitled, ''The Structure of Papain Refined at 1.65 A Resoltion'', Kamphuis et al. discovered interesting information on <scene name='Sandbox_34/All_bonding_shenanigans/1'>direct protein-protein contacts</scene> between molecules of papain in solution. These contacts, communicated in Table 7 of their paper, consist of nine hydrogen-bond connections and three ionic interactions. The strongest salt bridge exists between <scene name='Sandbox_34/Arg191asp140intraxn/2'>asparagine-140 and arginine-191</scene>.<ref name="Structure" /> | |||
=== Solvent Interactions === | === Solvent Interactions === | ||
Papain binds both <scene name='Sandbox_34/Papainwithwaterandmtoh/1'>methanol and water molecules</scene> via hydrogen bonding. This solvent mixture of 62%, (w/w) methanol to water was used in order to obtain a C-type crystal. Papain has an interesting method of utilizing hydrogen bonding with <scene name='Sandbox_34/Papainwithwateronly/1'>water molecules</scene>, shown in cyan. A refined crystal structure of papain revealed that water forms something similar to a hydration shell around individual molecules of papain. The interaction of papain with these water molecules leads to less interaction between papain molecules, and contributes to the stability of the crystal structure of papain. The water molecules also form hydrogen bonds within the <scene name='Sandbox_34/Papainwithwaterandmtoh/2'>active site</scene>, providing even more stability for the crystalline structure.<ref name="Structure" /> | Papain binds both <scene name='Sandbox_34/Papainwithwaterandmtoh/1'>methanol and water molecules</scene> via hydrogen bonding. This solvent mixture of 62%, (w/w) methanol to water was used in order to obtain a C-type crystal. Papain has an interesting method of utilizing hydrogen bonding with <scene name='Sandbox_34/Papainwithwateronly/1'>water molecules</scene>, shown in cyan. A refined crystal structure of papain revealed that water forms something similar to a hydration shell around individual molecules of papain. The interaction of papain with these water molecules leads to less interaction between papain molecules, and contributes to the stability of the crystal structure of papain. The water molecules also form hydrogen bonds within the <scene name='Sandbox_34/Papainwithwaterandmtoh/2'>active site</scene>, providing even more stability for the crystalline structure.<ref name="Structure" /> | ||
[[Image:Papainmech6.jpg|275px|right|thumb| A general mechanism of papain catalysis<ref>[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref>.]] | |||
[[Image:Papainmech6.jpg| | |||
=== Specificity === | === Specificity === | ||
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The mechanism of cysteine proteases is very similar to that of serine proteases. However, instead of requiring a triad, papain only requires a diad. The sulfhydryl group on cysteine executes a nucleophilic attack on the peptide bond of the protein it wishes to cleave. Asparagine-175 keeps histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual mechanism. Opening up the carbonyl, the sulfhydryl group of CYS-25 is stabilized by HIS-159. As the carbonyl reforms, the peptide bond is broken, leaving the amide group to fend for itself.<ref>[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> | The mechanism of cysteine proteases is very similar to that of serine proteases. However, instead of requiring a triad, papain only requires a diad. The sulfhydryl group on cysteine executes a nucleophilic attack on the peptide bond of the protein it wishes to cleave. Asparagine-175 keeps histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual mechanism. Opening up the carbonyl, the sulfhydryl group of CYS-25 is stabilized by HIS-159. As the carbonyl reforms, the peptide bond is broken, leaving the amide group to fend for itself.<ref>[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> | ||
== Inhibitors == | == Inhibitors == | ||
