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| | + | ==Introduction to Adenylate Kinase== |
| | + | <Structure load='1ake' size='500' frame='true' align='right' caption='Adenylate Kinase' scene='Awesomeness' /> |
| | + | <scene name='Sandbox_34/Adenylate_kinase/1'>Adenylate Kinase</scene> is a very important protein in many biological processes, especially monitoring metabolism. |
| | + | Adenylate Kinase is made up of <scene name='Sandbox_34/Adenylate_kinase_colors/1'>9 beta sheets (light blue) and 9 alpha helices (dark blue).</scene> These are very important for the conformation and function of the enzyme. The beta sheets are parallel in the protein which puts extra strain on the <scene name='Sandbox_34/Adenylate_kinase_hbonds_yell/1'>hydrogen bonds (yellow)</scene> within the protein. That is why there appear to be angled in stead of straight (which is found in anti-parallel beta sheets). These bonds are also very important in that they help to hold the helices and sheets together. These hydrogen bonds are formed from interactions among the proteins many hydrogen containing residues. Without them the protein would unravel and denature. |
| | + | The <scene name='Sandbox_34/Adenylate_kinasehydrophobic_go/3'>hydrophobic interactions (deep pink)</scene> also play a huge role in holding this protein together. These interactions are formed by the hydrophobic or non-polar residues of the proteins. |
| | + | However, the hydrophobic interactions are not the only thing holding it together or helping to keep its conformation. There are also many <scene name='Sandbox_34/Adenylate_kinase_andpolarside/1'>polar and charged side groups (green)</scene> that help maintain the hydrophobic interactions and help stabilize the protein by interacting with one another. The interaction between the polar groups and the hydrophobic groups can be seen by the pink transparent hydrophobic groups and the green polar groups. |
| | + | ==Structual Properties of Adenylate Kinase== |
| | + | Proteins can be crystallized which can be very helpful for understanding the relative shape and sometimes functions of the protein. When proteins are crystallized they often have water molecules embedded in them because of how tightly and deep the water molecules can become wedged within the protein. The <scene name='Sandbox_34/Adenylate_kinase_water/1'>water molecule interactions (magenta)</scene> with the protein can be seen surrounding and inside some parts of the protein. The <scene name='Sandbox_34/Adenylate_kinase_h2o_interact/1'>hydrophilic (blue) and hydrophobic (green)</scene> parts of the protein act differently with the water molecules. As seen in the previous link, the hydrophilic parts interact with the water molecules while the hydrophobic parts of adenylate kinase stay away from them. Another view <scene name='Sandbox_34/Adenylate_kinase_waterextra/2'>here</scene> shows it transparently to better see the interactions. |
| | | | |
| - | == Papain ==
| + | Ligands on proteins vary greatly. The <scene name='Sandbox_34/Adenylate_kinase_ligand/1'>ligand and residue interactions</scene> of adenylate kinase are based on the non-hydrolysable ligand of the protein. The residues interacting with the ligand are polar and charged because this ligand is also polar and charged, it is therefore stabilized by the residues. Also the <scene name='Sandbox_34/Adenylate_kinase_activeresidue/1'>active site residues(yellow)</scene> are also interacting with the ligand. |
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| - | <Structure load='9pap' size='350' frame='true' align='right' caption='Papain' scene='Sandbox_34/Entire_protein_with_ligandscys/2' /> | + | |
| - | === Introduction ===
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| - | '''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.
| + | ==Conclusion== |
| - | | + | Adenylate Kinase is a very cool and important protein. Many of the internal interactions of the protein, whether hydrophobic or hydrophilic, are very important for understanding the function of this protein. These interactions also help us to understand why the conformation of the protein is the shape that it is. These are all very important for understanding the protein and how to use it in biological studies and treatments. |
| - | === 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" />
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| - | | + | |
| - | == Structure ==
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| - | <Structure load='9pap' size='375' frame='true' align='left' caption='Papain and Structure' scene='Sandbox_34/Subunitsrandl/3'/>
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| - | Papain is a relatively simple enzyme. It consists of only one chain of 212 residues, with a secondary structure composed of 21%
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| - | <scene name='Sandbox_34/Betasheets/1'>beta sheets</scene> and 25% <scene name='Sandbox_34/Alphahelices/1'>alpha helices</scene>.<ref name="RSCB PDB">http://www.rcsb.org/pdb/explore/explore.do?structureId=9PAP</ref> Its tertiary structure has three disulfide bonds, illustrated in yellow, between Cys22 and Cys63, Cys56, and Cys 95,and Cys153 and Cys200. The single chain is separated into <scene name='Sandbox_34/Subunitsrandl/1'>two domains</scene>: R in purple, and L in gray. A cleft is formed in which the <scene name='Sandbox_34/2subuwithactivesite/4'>active site</scene>, consisting of Cys25, His159, and Asp175, 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, illustrated in white, between both the <scene name='Sandbox_34/Pap_with_h-bonding_btwnbckbne/2'>backbone</scene> and the <scene name='Sandbox_34/Pap_with_h-bonding_btwnsdchns/3'>residues</scene> help coordinate papain's 3D conformation. <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" />
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| - | === Solvent Interactions ===
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| - | Papain binds both <scene name='Sandbox_34/Papainwithwaterandmtoh/1'>methanol and water molecules</scene> via hydrogen bonding that give stability to the papain crystalline structure. 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. The water molecules also form hydrogen bonds within the <scene name='Sandbox_34/Papainwithwaterandmtoh/2'>active site</scene>, providing even more stability for the structure.<ref name="Structure" />
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| - | [[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>.]]
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| - | === Specificity ===
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| - | 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.
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| - | === Catalytic Mechanism ===
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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>
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| - | == Inhibitors ==
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| - | There are many inhibitors for papain because of its broad specificity. It is often used as a model enzyme for those in the papain super-family, such as cathepsin L and cathepsin K. The inhibition of papain is usually due to active site restriction of cysteine-25 and histidine-159. The interest in developing inhibitors for the papain super-family lies with the desire to effectively inhibit other cysteine proteases that incur unfavorable effects within the body.
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| - | <Structure load='9pap' size='350' frame='true' align='left' caption='Papain and Inhibition' scene='Sandbox_34/9pap_active_site/1' />
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| - | ===Cathepsin L===
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| - | <scene name='Sandbox_34/Cathepsin_l/1'>Cathepsin L</scene> 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>
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| - | | + | |
| - | ===Cathepsin K===
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| - | The goal of research for the development of an inhibitor for <scene name='Sandbox_34/Cathepsink/1'>cathepsin K</scene> 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
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| - | <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>
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| - | ===Human Stefin B===
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| - | 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>
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| - | <scene name='Sandbox_34/Subunitsrandl/4'>Planar circles at the voronoi surface</scene>
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| - | == References ==
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| - | <references />
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| - | == External References ==
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| - | [http://en.wikipedia.org/wiki/Papain Wikipedia]
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is a very important protein in many biological processes, especially monitoring metabolism.
Adenylate Kinase is made up of These are very important for the conformation and function of the enzyme. The beta sheets are parallel in the protein which puts extra strain on the within the protein. That is why there appear to be angled in stead of straight (which is found in anti-parallel beta sheets). These bonds are also very important in that they help to hold the helices and sheets together. These hydrogen bonds are formed from interactions among the proteins many hydrogen containing residues. Without them the protein would unravel and denature.
The also play a huge role in holding this protein together. These interactions are formed by the hydrophobic or non-polar residues of the proteins.
However, the hydrophobic interactions are not the only thing holding it together or helping to keep its conformation. There are also many that help maintain the hydrophobic interactions and help stabilize the protein by interacting with one another. The interaction between the polar groups and the hydrophobic groups can be seen by the pink transparent hydrophobic groups and the green polar groups.
Proteins can be crystallized which can be very helpful for understanding the relative shape and sometimes functions of the protein. When proteins are crystallized they often have water molecules embedded in them because of how tightly and deep the water molecules can become wedged within the protein. The with the protein can be seen surrounding and inside some parts of the protein. The parts of the protein act differently with the water molecules. As seen in the previous link, the hydrophilic parts interact with the water molecules while the hydrophobic parts of adenylate kinase stay away from them. Another view shows it transparently to better see the interactions.
Ligands on proteins vary greatly. The of adenylate kinase are based on the non-hydrolysable ligand of the protein. The residues interacting with the ligand are polar and charged because this ligand is also polar and charged, it is therefore stabilized by the residues. Also the are also interacting with the ligand.
Adenylate Kinase is a very cool and important protein. Many of the internal interactions of the protein, whether hydrophobic or hydrophilic, are very important for understanding the function of this protein. These interactions also help us to understand why the conformation of the protein is the shape that it is. These are all very important for understanding the protein and how to use it in biological studies and treatments.
