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| | <!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | | <!-- PLEASE ADD YOUR CONTENT BELOW HERE --> |
| - | | + | <Structure load='1AKE' size='500' frame='true' align='right' caption='Adenylate Kinase' scene='Insert optional scene name here' /> |
| - | = '''Papain''' = | + | ==Adenylate Kinase== |
| - | | + | Adenylate kinase, also known as "ADK", is an enzyme which speeds up the reaction that includes the interconversion of adenine nucleotides. The protein's flexibility allows it to bind to certain substrates known as ligands. Adenylate kinase is known for influencing cellular energy homeostasis. |
| - | ==Introduction== | + | |
| - | ===Did you know?===
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| - | [[Image:Papain_cartoon.png|200px|left|thumb|Cartoon Peak at Papain]]
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| - | <scene name='Sandbox_35/Papain/1'>Papain</scene>. Meat tenderizer. Old time home remedy for insect, jellyfish, and stingray stings<ref>[http://www.ameriden.com/products/advanced-digestive-enzyme/] Ameridan International</ref>. Who would have thought that a sulfhydryl protease from the latex of the papaya fruit, ''Carica papaya'' and ''Vasconcellea cundinamarcensis'' would have such a practical application beyond proteopedia?
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| - | This protease belongs to an extended family of aminopeptidases, dipeptidyl peptidases, endopeptidases, and other enzymes having both exo- and endo-peptidase activity. The inactivated zymogen with N-terminal propeptide regions - providing stability in alkaline environments and enabling proper folding - is activated through removal of the propeptide regions. <ref>PMID: 7845226</ref> The protein is primarily secreted with its pro-region enabling transport from zymogen to lysosome through membrane association and mediation. <ref>PMID: 12188906</ref>
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| - | ===Historicity=== | + | |
| - | Papain made its first appearance in the ''Calcutta Medical Journal'' entitled “The Solvent Action of Papaya Juice on Nitrogenous Articles of Food” when G.C Roy was investigating the enzyme in 1873. In the late 19th century, Wurtz and Bouchut dubbed the partially purified enzyme "papain." <ref>Menard and Storer 1998</ref> At the time, it was viewed as proteolytically active constituent in the latex of tropical papaya fruit. <ref>Wurtz and Bouchut 1879</ref> As separation and purification techniques improved, pure papain was able to be isolated. In becoming the second enzyme to attain an X-ray crystallized structure and the first cysteine protease to behold an identifiable structure, papain fueled greater advances in enzymatic studies. <ref>PMID: 5681232</ref>
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| - | Papain. Lights. Camera. Action!
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| | ==Structure== | | ==Structure== |
| - | <StructureSection load='9pap' size='500' side='right' caption='Structure of HMG-CoA reductase (PDB entry [[9pap]])' scene=''>
| + | The <scene name='Sandbox_35/Secondary_structure_colored/1'>secondary structure</scene> of Adenylate Kinase has alpha helicies (purple) and beta sheets (dark pink) that circle around and enclose the non-hydrolysable part of the protein,(seen in the center). This is known as the ligand, which does not experience hydrolysis. These alpha helicies and beta sheets are known as the "backbone" of the protein. |
| - | ====Structural Elements====
| + | One of the main components of protein structure are the <scene name='Sandbox_35/Secondary_h_bonds2/1'>hydrogen bonds</scene> which are shown in green on this structure. The hydrogen bonds form links between adjacent amino acids, contributing to the protein structure and fold. Hydrogen bonding is an excellent stabilizing factor for the protein! The hydrogen bonds on the beta sheets are in an anti-parallel configuration, which offers stability for the protein. |
| - | Papain's single polypeptide chain consists of 212 amino acid residues which fold to form a groove containing the active site between its two domains. Its <scene name='Sandbox_35/Secondary_structure_papain/2'>secondary structure</scene> consists of 17 <scene name='Sandbox_35/2nd_struc_papain_beta/2'>beta sheet</scene> strands and 7 <scene name='Sandbox_35/2nd_struc_papain_helix/2'>alpha helices</scene> giving it a composition 21% and 25% respectively. <ref name="9PAP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP]9PAP PDB</ref> The hydrogen bonds within the alpha helices are shorter than the typical alpha helix because of C=O being directed further away from the helical axis. Moreover, the beta sheet hydrogen bonding constraints and structural angles show great variation; hydrogen bonds in the sheets' central tend to be shorter than on the fringes. Three disulfide bonds, like <scene name='Sandbox_35/Papain_cys_bond/1'>Cys 22-Cys 63</scene>, serve to hold papain's tertiary structure together. <ref name="Kamphuis">PMID: 6502713</ref>
| + | ==Hydrophobic and Hydrophilic Residue Composition== |
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| + | Adenylate kinase is composed of both hydrophobic and hydrophilic residues, and folds accordingly to obtain the optimum environments for the nature of both kinds of residues. The <scene name='Sandbox_35/Secondary_hydrophobs_only_gre/1'>hydrophobic residues</scene> shown in grey are buried within the folded protein, away from contact with the solvent. This action represents the hydrophobic effect taking place, which is mainly driven by entropy. Surrounding the outside of the protein are the <scene name='Sandbox_35/Secondary_hphobic_and_hphilic/1'>hydrophilic residues</scene> (dark green). These residues are polar and can be in caontact with the surrounding solvent in the protein's environment. These residues can either be charged or uncharged, can hydrogen bond with water, and are generally more soluble. They cover the hydrophobic amino acid residues to protect them from the solvent. |
| - | [[Image:Domains of Papain.png|200px|left|thumb|Contacts between Papain Subunits.<ref name="Richardson">[http://kinemage.biochem.duke.edu/teaching/anatax/html/anatax.2i.html] Jane S. Richardson</ref>.]]
| + | ==Water and Solvent== |
| - | | + | Water is very important when it comes to protein folding and structure. It determines the conformation of exposed side chains, stabilizes the ends of secondary structures, and occupy positions at active sites where they influence substrate binding and sometimes catalysis. Adenylate kinase in <scene name='Sandbox_35/Secondary_with_water_molecules/1'>solvent</scene> is mostly surrounded by water molecules around the exterior area of the protein; However, it also can utilizes the water molecules to increase efficent substrate binding. The water molecules (light blue) surround the outside of the protein, interacting with the polar hydrophilic residues. However, some water molecules are seen in contact with the ligand (light green center) where the molecules are influencing catalysis. |
| - | | + | ==Adenylate Kinase and The Ligand== |
| - | Papain's two subunits are held together with "arm" linkage where one end of the protein chain holds the opposite domain. In papain's case the "arm crossing" primarily occurs on or near the surface. <ref name="Richardson" />
| + | The ligand(dark pink) in the center of the protein has specific residues surrounding it that are also known as the <scene name='Sandbox_35/Secondary_with_ligand_in_cente/1'>ligand contacts</scene>. These residues have polar-charged side chains, which stabilize the ligand. The ligand in Adenylate kinase is a molecule which is able to bind to the protein's specific active site. |
| - | | + | ==Catalytic Residues== |
| - | | + | The <scene name='Sandbox_35/Secondary_catalytic_residues/1'>catalytic residues</scene> (black) are found in the center of the protein, lining the active site where the ligand binds. These residues, also known as "active site residues", help with recognition of the ligand. The ligand binds with the protein in various ways: hydrogen bonds, hydrophobic interactions, temporary covalent interactions, or a mixture of the mentioned methods. The catalytic residues assist the reaction by acting as proton donors or acceptors. In the big picture, all of this helps the enzyme protein lower the activation energy of the ligand to speed up the reaction efficiently. |
| - | ====Active Site==== | + | |
| - | Located in the cleft between its domains, the active site consists of seven subsites (S1-S4 and S1’-S3’) each accommodating one amino acid residue of a substrate (P1-P4 and P1’-P3’). <ref>Schechter and Berger 1967</ref> The specificity of the active site is controlled by the S2 subsite which is a hydrophobic pocket that accommodates the P2 side chain of the substrate. Particularly at this subsite, papain shows specific substrate preferences for bulky hydrophobic or aromatic residues. Outside of the S2 subsite preferences, the active site appears to lack clearly defined residue selectivity from within its site. <ref>Kimmel and Smith 1954</ref>
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| - | The <scene name='Sandbox_35/Active_site_papain/4'>active site</scene> primarily consist of three main residues Cys 25, His 159, and Asn 175 holding resemblance to the catalytic triad of chymotrypsin <ref name="Wang">PMID: 8140097</ref><ref>PMID: 2397208</ref>. However, growing studies are showing that the mechanism behind catalysis may actually involve a double catalytic site - consisting of Cys 25- His 159- Asn 175 ''and'' Cys 25- His 159-<scene name='Sandbox_35/Active_site_papain/5'>Asp 158</scene>! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." <ref name="Wang" /> In addition, replacement of Asn 175 with other residues such as Ala mutants, reveals a decrease in kcat revealing less efficiency. Despite this, the rate of hydrolysis is still significantly larger than non-catalytic rates, suggesting a less essential role Asn 175 plays than originally thought. Building on these observations, alteration to the 175 side chain results in less thermal stability lending thought that Asn 175 plays a more structural rather than catalytic role. <ref>[http://www.jbc.org/content/270/28/16645.abstract] The Journal of Biological Chemistry </ref> | + | |
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| - | Crystallization of the protease under conditions of 62% (w/w) methanol in water reveals water playing a crucial role in providing structural stability. The 21 internal water molecules surrounding adjacent papain molecules appear to form an encasement that limit protein to protein interaction. <ref name="Kamphuis" />
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| - | ====Distribution of Residues==== | + | |
| - | Although papain has a scattered distribution of <scene name='Sandbox_35/Papain_acid_and_basic_residues/1'>acidic and basic residues</scene>, it can be seen to have more basic residues than acidic, shedding understanding into the application of its use as a digestive supplement. <ref>[http://www.webmd.com/vitamins-supplements/ingredientmono-69-PAPAIN.aspx?activeIngredientId=69&activeIngredientName=PAPAIN] WebMD</ref> Seeing its <scene name='Sandbox_35/Hydrophobicity_papain/3'>polar and non-polar residues</scene> further shows <scene name='Sandbox_35/Papain_polar/1'>polar residues</scene> remaining mostly on the exterior while <scene name='Sandbox_35/Nonpolar_papain/2'>non-polar residues</scene> sequestering near the center. Observations have revealed that the proteins atomic positions are more ordered going from outside toward the center and also disclose the hydrophobic core of the enzyme. <ref name="Kamphuis" />
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| - | ====Ligand interactions and Pseudo Substrates==== | + | |
| - | Papain is said to have 29 identifiable methanol molecules that encircle around it as <scene name='Sandbox_35/Papain_ligand/1'>ligands</scene>. The polarity of the ligands result in hydrogen bonding interactions, possibly providing further stability for papain structure. <ref name="Kamphuis" />
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| - | <scene name='Sandbox_35/Cathepsin_l_specific_inhibitor/3'>Cathepsin L specific inhibitor</scene> is part of a series known as CLIK inhibitors and was used on papain as an assessment of inhibition specificity for cathepsin enzymes. Structural differences between Papain-CLIK 148 complex and original papain is not very drastic. Minute changes result primarily from alterations in surface proteins except where a covalent bond is formed between the C2 on <scene name='Sandbox_35/Clik_cys/1'>CLIK 148 and Cys 25 residue</scene>. The primary <scene name='Sandbox_35/Cathepsin_interaction/3'>interactions</scene> between pseudo-substrate/inhibitor and papain were non-water hydrogen bonds and mostly hydrophobic interactions. CLIK 148's binding to the active site of papain is in a non-substrate mode with the main site showing pyrimidine ring interaction between <scene name='Sandbox_35/Clik_trp_177/1'>Trp 177 and CLIK 148</scene>. Hydrogen bonding is observed between the oxygens in <scene name='Sandbox_35/Clik_gly_gln/1'>CLIK 148 to Gln 19 and Gly 66 residues</scene>. Moreover, a water molecule has been observed to be near the His 159 residue enabling greater hydrogen bonding, once again highlighting solvents role in stability. <ref>PMID: 10600517</ref> Through modeling, more favorable energetics has also been revealed when hydrophobic and aromatic parts of the ligand occupy the S2, S3, and S1' subsites with at least three hydrogen bonding contacts between the protein conserved binding site residues and the ligand. <ref>PMID: 9472614</ref>
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| - | </StructureSection>
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| - | ==Catalytic Mechanism==
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| - | [[Image:Papainmech6.jpg|200px|left|thumb| General mechanism of papain catalysis<ref name="Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref>.]]
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| - | Papain's catalytic mechanism is like serine proteases. Its catalytic triad of residues Cys 25- His159- Asn-175 appear to work with a fourth residue, Gln-19, suspected to be involved in oxyanion hole formation. When a peptide binds to the active site, His-159 deprotonates Cys-25 which in turn attacks the substrate carbonyl carbon. The oxyanion hole then stabilizes the resulting covalent, tetrahedral intermediate. Subsequently, nitrogen in the peptide bond is protonated by His-159 (acting as an acid). This action frees the C-terminal portion of the peptide so that it is released. The entrance of water into the active site then attacks the carbonyl carbon while it is deprotonated by His-159, resulting in another tetrahedral covalent intermediate once again stabilized through the oxyanion hole. At the end, carbonyl reformation and the Cys-25 sulfur action as the leaving group releases the N-terminal portion of the peptide. The enzyme is regenerated for the cycle to begin again. <ref name="Maine" />
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| - | ==Other Interaction with Inhibitors and Effectors==
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| - | [[Image:Papain Simple Cleavage.jpg|200px|right|thumb|Simple Overview of Cleavage by Papain. <ref>[http://www.worthington-biochem.com/pap/default.html] Worthington Biochemical Corporation </ref>]]
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| - | Except for valine, papain prefers to cleave at hydrophobic residues alanine, leucine, isoleucine, phenylalanine, tryptophan, or tyrosine <ref>[http://www.sigmaaldrich.com/life-science/biochemicals/biochemical-products.html?TablePage=16410606] Sigma Aldrich Papain</ref>. Because of the importance of the oxyanion hole formation and the nucleophilic attack of cysteine, substances like cysteine, sulfide/sulfite, heavy metal chelating agents like EDTA, and N-bromosuccinimide behave as activators of the enzyme while PMSF, Hg2+ and other heavy metals, cystatin, leupeptin, sulfhydryl binding agents, carbonyl reagents, and alkylating agents serve as inhibitors. <ref>PMID: 6388564 </ref><ref>[http://www.biozym.de/datasheets/papain.php] Biozym </ref>
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| - | ==Fun Trivia==
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| - | Remember the 2002 SARS (Severe Acute Respiratory Syndrome) epidemic that placed global health, particularly in Southeast Asia, in a precarious state? On-going research is happening to further understand the mechanisms of this coronavirus, so that future steps can be taken for prevention. Its been found that the replication of RNA for this virus is mediated by two viral proteases that have many papain-like characteristics. <ref>PMID: 16306590 </ref>
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| - | Despite a low percentage of sequence identities, inhibition and sequence analyses have increasingly been drawing parallels between L proteinases, that involve the foot-and-mouth disease virus and equine rhinovirus 1, and papain. With a similar overall fold to papain and identifiable regions that resemble the five alpha-helices and seven beta-sheets of papain, L proteinases of foot-and-mouth disease virus and of equine rhinovirus 1 reveal a mode of operation that is very papain like. <ref>PMID: 9472614 </ref>
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| - | ==References==
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| - | <references />
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| - | <ref group="xtra">PMID:8140097</ref>
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Adenylate kinase, also known as "ADK", is an enzyme which speeds up the reaction that includes the interconversion of adenine nucleotides. The protein's flexibility allows it to bind to certain substrates known as ligands. Adenylate kinase is known for influencing cellular energy homeostasis.
The of Adenylate Kinase has alpha helicies (purple) and beta sheets (dark pink) that circle around and enclose the non-hydrolysable part of the protein,(seen in the center). This is known as the ligand, which does not experience hydrolysis. These alpha helicies and beta sheets are known as the "backbone" of the protein.
One of the main components of protein structure are the which are shown in green on this structure. The hydrogen bonds form links between adjacent amino acids, contributing to the protein structure and fold. Hydrogen bonding is an excellent stabilizing factor for the protein! The hydrogen bonds on the beta sheets are in an anti-parallel configuration, which offers stability for the protein.
Adenylate kinase is composed of both hydrophobic and hydrophilic residues, and folds accordingly to obtain the optimum environments for the nature of both kinds of residues. The shown in grey are buried within the folded protein, away from contact with the solvent. This action represents the hydrophobic effect taking place, which is mainly driven by entropy. Surrounding the outside of the protein are the (dark green). These residues are polar and can be in caontact with the surrounding solvent in the protein's environment. These residues can either be charged or uncharged, can hydrogen bond with water, and are generally more soluble. They cover the hydrophobic amino acid residues to protect them from the solvent.
Water is very important when it comes to protein folding and structure. It determines the conformation of exposed side chains, stabilizes the ends of secondary structures, and occupy positions at active sites where they influence substrate binding and sometimes catalysis. Adenylate kinase in is mostly surrounded by water molecules around the exterior area of the protein; However, it also can utilizes the water molecules to increase efficent substrate binding. The water molecules (light blue) surround the outside of the protein, interacting with the polar hydrophilic residues. However, some water molecules are seen in contact with the ligand (light green center) where the molecules are influencing catalysis.
The ligand(dark pink) in the center of the protein has specific residues surrounding it that are also known as the . These residues have polar-charged side chains, which stabilize the ligand. The ligand in Adenylate kinase is a molecule which is able to bind to the protein's specific active site.
The (black) are found in the center of the protein, lining the active site where the ligand binds. These residues, also known as "active site residues", help with recognition of the ligand. The ligand binds with the protein in various ways: hydrogen bonds, hydrophobic interactions, temporary covalent interactions, or a mixture of the mentioned methods. The catalytic residues assist the reaction by acting as proton donors or acceptors. In the big picture, all of this helps the enzyme protein lower the activation energy of the ligand to speed up the reaction efficiently.
