Sandbox 34: Difference between revisions
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=== History === | === History === | ||
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='390' frame='true' align='left' caption='Papain and Structure' scene='Sandbox_34/Subunitsrandl/3'/> | |||
<Structure load='9pap' size=' | |||
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" /> A <scene name='Sandbox_34/Rainbown-c/1'>Rainbow-C</scene> illustration of this shows, from the N-terminus in blue to the C-terminus in red, an easy means by which to track the residues through the molecule. 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" /> A <scene name='Sandbox_34/Rainbown-c/1'>Rainbow-C</scene> illustration of this shows, from the N-terminus in blue to the C-terminus in red, an easy means by which to track the residues through the molecule. 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. | <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. | ||
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" /> | |||
[[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|275px|right|thumb| A general mechanism of papain catalysis<ref>[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref>.]] | ||
=== Specificity === | === Specificity === | ||
Functioning as either an endopeptidase, an amidase, or an esterase, papain functions with a very broad specificity.<ref>http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0004197</ref>. It prefers amino acids that bear large hydrophobic side chains at the P2 position, and will not accept valine at the P1' position. <ref name="UniProt" /> Given its broad specificity, papain serves as a cheap and available cysteine protease which can be readily utilized by researchers as a prime example of the mechanisms of inhibition of other enzymes within the cysteine protease super-family. | Functioning as either an endopeptidase, an amidase, or an esterase, papain functions with a very broad specificity.<ref>http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0004197</ref>. It prefers amino acids that bear large hydrophobic side chains at the P2 position, and will not accept valine at the P1' position. <ref name="UniProt" /> Given its broad specificity, papain serves as a cheap and available cysteine protease which can be readily utilized by researchers as a prime example of the mechanisms of inhibition of other enzymes within the cysteine protease super-family. | ||
=== Catalytic Mechanism === | === Catalytic Mechanism === | ||
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> | ||
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<Structure load='9pap' size='350' frame='true' align='left' caption='Papain and Inhibition' scene='Sandbox_34/9pap_active_site/1' /> | <Structure load='9pap' size='350' frame='true' align='left' caption='Papain and Inhibition' scene='Sandbox_34/9pap_active_site/1' /> | ||
===Cathepsin L=== | ===Cathepsin L=== | ||
Cathepsin L is an endosomal cysteine protease that is believed to have both physiological and pathophysiological effects on the human body. It has been indicated not only in cancer, rhematoid arthritis, and osteo-arthritis, but its mechanism also appears similar to that of Ebola, SARS, and Leishmania. Understanding the mechanism of inhibition through the use of papain is therefore crucial to developing treatments for such diseases.<ref> PMID:18499453 </ref> An interesting inhibitor for cathepsin L developed using papain as the model protease is that of <scene name='Sandbox_34/Clik148_inhibitor/2'>Clik-148</scene>.<ref> PMID:10600517 </ref> It forms a <scene name='Sandbox_34/Clik148_inhibit_cys25/1'>covalent ligand-bound cysteine protease complex</scene> with Cys25. Five other residues are also involved in the bonding of Clik-148 to papain: Gln19, Gly66, Asp158, Trp177, and Ser205. These participate in hydrophobic, <scene name='Sandbox_34/Clik148ringstacking/2'>aromatic ring-stacking</scene>, and hydrogen bonding that effectively fill up the cleft between the two domains of papain.<ref> PMID:18598021 </ref> | Cathepsin L is an endosomal cysteine protease that is believed to have both physiological and pathophysiological effects on the human body. It has been indicated not only in cancer, rhematoid arthritis, and osteo-arthritis, but its mechanism also appears similar to that of Ebola, SARS, and Leishmania. Understanding the mechanism of inhibition through the use of papain is therefore crucial to developing treatments for such diseases.<ref> PMID:18499453 </ref> An interesting inhibitor for cathepsin L developed using papain as the model protease is that of <scene name='Sandbox_34/Clik148_inhibitor/2'>Clik-148</scene>.<ref> PMID:10600517 </ref> It forms a <scene name='Sandbox_34/Clik148_inhibit_cys25/1'>covalent ligand-bound cysteine protease complex</scene> with Cys25. Five other residues are also involved in the bonding of Clik-148 to papain: Gln19, Gly66, Asp158, Trp177, and Ser205. These participate in hydrophobic, <scene name='Sandbox_34/Clik148ringstacking/2'>aromatic ring-stacking</scene>, and hydrogen bonding that effectively fill up the cleft between the two domains of papain.<ref> PMID:18598021 </ref> | ||
===Cathepsin K=== | ===Cathepsin K=== | ||
The goal of research for the development of an inhibitor for cathepsin K is the hope to develop a treatment for osteoporosis. In two different cathepsin K inhibitors, referenced PDB codes <scene name='Sandbox_34/Cathkaldinhibit/1'>1BP4</scene> and <scene name='Sandbox_34/Cathkketoinhibition/1'>1BQI</scene>, it is evident that the inhibitor binds with much closer proximity than that of Clik148. 1BP4 is a cathepsin K inhibitor, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, that inhibits by interacting with 11 different residues on papain: Gln19, Gly20, Ser21, Gly23, Asn64, Gly65, Gln142, Asp158, His159, Trp177, and Trp181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to <scene name='Sandbox_34/Cathkaldinhibitpistacking/2'>ring stacking</scene> between the aromatic ring of the carbobenzyl group on 1BP4, and TRP177 of papain. The inhibition of papain by IBQI, carbobenzyloxy-(L)-leucinyl-(L)leucinyl methoxymethylketone, is quite similar to that of IBP4, although it does not bind quite as tightly. It binds to seven residues of papain: Gln19, Gly23, Gly65, Gln142, His159, Trp177, Trp181. Additionally, it has similar | The goal of research for the development of an inhibitor for cathepsin K is the hope to develop a treatment for osteoporosis. In two different cathepsin K inhibitors, referenced PDB codes <scene name='Sandbox_34/Cathkaldinhibit/1'>1BP4</scene> and <scene name='Sandbox_34/Cathkketoinhibition/1'>1BQI</scene>, it is evident that the inhibitor binds with much closer proximity than that of Clik148. 1BP4 is a cathepsin K inhibitor, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, that inhibits by interacting with 11 different residues on papain: Gln19, Gly20, Ser21, Gly23, Asn64, Gly65, Gln142, Asp158, His159, Trp177, and Trp181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to <scene name='Sandbox_34/Cathkaldinhibitpistacking/2'>ring stacking</scene> between the aromatic ring of the carbobenzyl group on 1BP4, and TRP177 of papain. The inhibition of papain by IBQI, carbobenzyloxy-(L)-leucinyl-(L)leucinyl methoxymethylketone, is quite similar to that of IBP4, although it does not bind quite as tightly. It binds to seven residues of papain: Gln19, Gly23, Gly65, Gln142, His159, Trp177, Trp181. Additionally, it has similar | ||
<scene name='Sandbox_34/Cathkketoinhibitionringstackin/2'>ring-stacking</scene> between the Cbz ring on the inhibitor and Trp 177, though it is more difficult to visualize with the given PDB file.<ref> PMID:9804696 </ref> | <scene name='Sandbox_34/Cathkketoinhibitionringstackin/2'>ring-stacking</scene> between the Cbz ring on the inhibitor and Trp 177, though it is more difficult to visualize with the given PDB file.<ref> PMID:9804696 </ref> | ||
===Human Stefin B=== | ===Human Stefin B=== | ||
Other inhibitors, such as <scene name='Sandbox_34/Pap_1stf/1'>human stefin B</scene>, illustrated in magenta, are much more complex in their <scene name='Sandbox_34/Pap_1stf/5'>inhibition</scene>. The human stefin B molecule has a five stranded beta-sheet that wraps around a five turn alpha-helix. The interface between human stefin B and papain is very tightly packed with 16% of stefin B becoming embedded within papain. A total of 128 <scene name='Sandbox_34/Pap_1stf/6'>intermolecular atom-atom interactions</scene> <4 A occur within the cleft in papain, although only CYS25 interacts with the inhibitor. In this figure, the residues of interaction for stefin B are shown in red, the residues of interaction for papain are shown in blue, and the residues of papain's active site are shown in green. Through this study, Stubbs et al. were able to conclude that cysteine proteinase inhibitors are "fundamentally different to [those] observed for serine proteinase inhibitors."<ref>PMID:2347312</ref> | Other inhibitors, such as <scene name='Sandbox_34/Pap_1stf/1'>human stefin B</scene>, illustrated in magenta, are much more complex in their <scene name='Sandbox_34/Pap_1stf/5'>inhibition</scene>. The human stefin B molecule has a five stranded beta-sheet that wraps around a five turn alpha-helix. The interface between human stefin B and papain is very tightly packed with 16% of stefin B becoming embedded within papain. A total of 128 <scene name='Sandbox_34/Pap_1stf/6'>intermolecular atom-atom interactions</scene> <4 A occur within the cleft in papain, although only CYS25 interacts with the inhibitor. In this figure, the residues of interaction for stefin B are shown in red, the residues of interaction for papain are shown in blue, and the residues of papain's active site are shown in green. Through this study, Stubbs et al. were able to conclude that cysteine proteinase inhibitors are "fundamentally different to [those] observed for serine proteinase inhibitors."<ref>PMID:2347312</ref> | ||
