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| - | <!-- PLEASE DO NOT DELETE THIS TEMPLATE --> | + | <Structure load='1ake' size='500' frame='true' align='right' caption='Adenylate Kinase' scene='Insert optional scene name here' /><!-- PLEASE DO NOT DELETE THIS TEMPLATE --> |
| | {{Template:Oberholser_Sandbox_Reservation}} | | {{Template:Oberholser_Sandbox_Reservation}} |
| - | =='''Introduction'''==
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| - | <applet load='9PAP' size='500' frame='true' align='right' scene='Sandbox_32/Papain/1' caption='Papain' /> | + | |
| - | Papain is a sulfhydryl protease from papaya latex (''Carica papaya'') and is used in food and pharmaceutical industries (7). It's PDB ID is 9PAP and it has an E.C. # of 3.4.22.2. It is composed of a single amino acid chain. The structure shown is of 1.65Å resolution (3).
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| - | =='''Structure'''== | + | == '''Adenylate Kinase''' (PDB ID #: 1ake)== |
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| - | Papain contains 212 amino acid resiudes and a molecular weight of 23.4 kDa (6). It's secondary structure contains 7 <scene name='Sandbox_32/Alpha_helix/3'>alpha helices</scene> that make up 25% of the protein, and 17 <scene name='Sandbox_32/Beta_sheet/4'>beta sheets</scene> making up about 17% (5).
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| - | In its 3D structure, Papain is stabilized by many <scene name='Sandbox_32/Hydrophobic_residues/2'>hydrophobic interactions</scene> These interactions have been shown to be very important in the overall structure and folding of the protein (7). The <scene name='Sandbox_32/Negatively_charged_residues/1'> charged residues </scene> are also shown along with both <scene name='Sandbox_32/Hydrophobic/1'>hydrophobic and charged residues </scene>. In addition, papain is made up of <scene name='Sandbox_32/Domains_l_and_r/2'>two domains</scene>, L and R (L=green and R=blue). The L domain contains alpha-helical structural components (residues 10-111, 208-212) while the R domain (residues 1-9, 112-207) is characterized by anti-parallel beta sheet structure (7). In addition to these interactions, the overall structure of papain is stabilized by three <scene name='Sandbox_32/Disulfide_bonds/4'>disulide bonds</scene>. There are 6 cysteine residues that make up these bonds--Cys 22-63, 56-95, 153-200 (5).
| + | The <scene name='Sandbox_32/Chain_a/2'>A Chain</scene> by itself may be in a slightly different conformation than when it is <scene name='Sandbox_32/Both_chains/1'>attached</scene> to the B chain (as found in nature). |
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| - | =='''Catalytic Mechanism'''==
| + | Adenylate Kinase contains both types of secondary structure, <scene name='Sandbox_32/Helices_sheets/2'>alpha helices and beta sheets</scene>. In this scene, alpha helices are in light blue and beta sheets are in yellow. |
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| - | As a sulfhydryl protease, Papain has a catalytic site with three important residues --Cysteine-25, Histidine-159,and Asparganine176 (4). The catalytic site is located in the <scene name='Sandbox_32/Binding_cleft/2'>binding cleft</scene> between the L and R domains of the protein The <scene name='Sandbox_32/Catalytic_site/1'>catalytic site</scene> centers around Cysteine-25 as it is a sulfhydryl protease. Sulfur on the cysteine will nucelophilically attack its substrate. Histidine acts to deprotonate sulfur (Cys-25), allowing it to be negatively charged. Once sulfur attacks the peptide,the tetrahedral intermediate is stabilized by a proposed oxyanion hole, specifically by asparganine (Asn-176) (1). The charged imidazolium ion of papain will in turn protanate the nitrogen in the peptide bond, releasing the C-terminal part of the peptide (5). Water enters the active site and ultimately regenerates the enzyme and allowing for the release of the N-terminal portion of the cleaved peptide.
| + | The <scene name='Sandbox_32/H_bonds_2/1'>hydrogen bonding</scene> highlighted in this scene shows us that the secondary structure (helices and sheets) is held together by hydrogen bonds. The beta sheets appear to be parallel, as the H-bonds are not all aligned in one direction. |
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| - | Papain is very non-specific in the type of peptide bonds that it cleaves. It will cleave bonds of most basic amino acids. It will also cleave after leucine or glycine residues and will hydrolyze esters and amides (3).
| + | <scene name='Sandbox_32/Hydrophobic_stickandwireframe/1'>Hydrophobic side chains</scene>, highlighted here in pink, tend to point towards the inside of the molecule where they do not have to interact with the polar water molecules. |
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| - | =='''Inhibition'''==
| + | The <scene name='Sandbox_32/Hydrophilic/1'>hydrophilic side chains</scene>, highlighted here in blue along with the transparent pink hydrophobic residues, tend to be pointed towards the outside of the protein, where it will interact with the cytosol. |
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| - | Papain is inactivated by oxygen iodine, hydrogen peroxide, and EDTA (4). Also, it is is irreversibly inhibited by N-ethylmaleimide and iodoacetate. Other inhibitors of Papain are <scene name='Sandbox_32/Inhibition/1'>ICP</scene> (inhibitor of cysteine protease) and <scene name='Sandbox_32/Khq/1'>ZLFG-DAM </scene> (1). With ZLFG-DAM(diazomethylketone), the methylene carbon will covalently bind to the cysteine-25 carbon and the hydrophobic pocket near the active site will also be inhibited (1).
| + | <scene name='Sandbox_32/Water_ligand/3'>Water molecules</scene> (shown in blue) surround and solvate the protein. The ligand is highlighted in green. The waters seem to congregated on one side than the other, possibly to make room for chain B to bind. |
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| - | This scene should show the contacts between papain and its inhibitor <scene name='Sandbox_32/Inhibitor/1'>ZLFG-DAM</scene>
| + | Sidechain and ligand <scene name='Sandbox_32/Sidechain_ligand_interaction/2'>interactions</scene> are shown in this scene. The ligand is in orange, and the interacting side chains are in dark blue and red. Of these contacting residues, only some actually catalyze the reaction on the substrate. These <scene name='Sandbox_32/Active_site_2/1'>active site</scene> residues are highlighted in red. These are the residues which interact chemically with the substrate to turn it into product. The non-active site residues are important in substrate (or ligand) binding. |
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| - | Water and methanol can be considered other ligands to Papain. When obtaining the crystal structure of the enzyme, the solvent becomes part of the stabilized structure. Another ligand that can be considered for Papain is water. Water molecules play an important role in maintaining structural stability. There are 21 water molecules that are located in contact areas between adjacent papain molecule (2).
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| - | =='''References'''==
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| - | 1. Alphey, Magnus S. & Hunter, William N. (2006). High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei. Crystallography, 62, 504-508.
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| - | 2. Janowski, R., Kozak, M., Jankowska, E., Grzonka, Z., & Jaskolski, M (2004). Two polymorphs of a covalent complex between papain and a diazomethylketone inhibitor. J.Pept. Res. 64, 141-150.
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| - | 3. Kamphuis, I. G., Kalk, K.H., Swarte, M.B., & Drenth, J (1984). Structure of papain refined at 1.65 A resolution. J. Mol. Biol. 179, 233-56.
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| - | 4. Lowe, G. The structure and mechanism of action of papain (1970). Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 257, 237-248.
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| - | 5. PDB (2011). Retrieved November 10, 2011 from http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP
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| - | 6. PDBsum (2011). Retrieve November 12, 2011 from http://www.ebi.ac.uk/pdbsum/9pap
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| - | 7. Sathish, Hasige A., Kumar, Parigi Ramesh, & Prakash, Vishweshwaraiah (2009). The differential stability of the left and right domains of papain. Process Biochemistry. 44, 710-16.
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The by itself may be in a slightly different conformation than when it is to the B chain (as found in nature).
Adenylate Kinase contains both types of secondary structure, . In this scene, alpha helices are in light blue and beta sheets are in yellow.
The highlighted in this scene shows us that the secondary structure (helices and sheets) is held together by hydrogen bonds. The beta sheets appear to be parallel, as the H-bonds are not all aligned in one direction.
, highlighted here in pink, tend to point towards the inside of the molecule where they do not have to interact with the polar water molecules.
The , highlighted here in blue along with the transparent pink hydrophobic residues, tend to be pointed towards the outside of the protein, where it will interact with the cytosol.
(shown in blue) surround and solvate the protein. The ligand is highlighted in green. The waters seem to congregated on one side than the other, possibly to make room for chain B to bind.
Sidechain and ligand are shown in this scene. The ligand is in orange, and the interacting side chains are in dark blue and red. Of these contacting residues, only some actually catalyze the reaction on the substrate. These residues are highlighted in red. These are the residues which interact chemically with the substrate to turn it into product. The non-active site residues are important in substrate (or ligand) binding.
