Sandbox 32

<!-- PLEASE DO NOT DELETE THIS TEMPLATE -->

{{Template:Oberholser_Sandbox_Reservation}}

=='''Introduction'''==

<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).

=='''Structure'''==

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).

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).

=='''Catalytic Mechanism'''==

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.

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).

=='''Inhibition'''==

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).

This scene should show the contacts between papain and its inhibitor <scene name='Sandbox_32/Inhibitor/1'>ZLFG-DAM</scene>

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).

=='''References'''==

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.

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.

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.

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.

5. PDB (2011). Retrieved November 10, 2011 from http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP

6. PDBsum (2011). Retrieve November 12, 2011 from http://www.ebi.ac.uk/pdbsum/9pap

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.