Sandbox 36

Please do NOT make changes to this Sandbox. Sandboxes 30-60 are reserved for use by Biochemistry 410 & 412 at Messiah College taught by Dr. Hannah Tims during Fall 2012 and Spring 2013. spinqualitylabelsall models Papain (9PAP) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Papain (finished) Introduction Papain is a sulfhydryl or cysteine protease derived from the papaya fruit. Papain has many varied and important commercial uses. It is often used as a meat tenderizer because it can hydrolyze the peptide bonds of collagen, elastin, and actomyosin. It is also used in contact lens solution to remove protein deposits on the lenses. Papain also has many medical uses and is used to treat pain, swelling, and fluid retention following trauma and surgery. More commonly, papain is used as a digestive supplement. Structure Papain consists of a single polypeptide chain of 212 amino acid residues split into two lobes. 55 of these residues form 7 helices and 45 residues form 17 beta sheet strands. Besides these structures, the secondary structure of papain is irregular. As with all proteins, folding to form secondary and tertiary structures is largely determined by the interactions of the hydrophobic residues to exclude water (hydrophobic residues shown in purple). A majority of these hydrophobic residues form hydrophobic cores within each of the lobes ensuring their stability (surface residues are transparent, buried hydrophobic residues are opaque and colored). The remaining residues are polar, some carrying a negative charge (acidic) at physiological pH, others a positive charge (basic), the rest of the polar residues are neutral. The protein's tertiary structure consists of two domains divided by a cleft in which the active site resides. Papain has several methanol ligands associated with it. Various residues on papain hydrogen bond to these methanol molecules (methanol molecules are shown in green and associated residues are shown in blue). Active Site and Catalytic Diad The 212 residues of Papain can be split nearly in half to produce two domains though the enzyme consists only of one polypeptide chain. The active site is located in the cleft between the two domains. The two domains interact with one another via hydrophobic interactions, hydrogen bonds, and electrostatic interactions in this cleft. For example, Valine-32 from the L Domain hydrophobically interacts with the carbon atoms on residues Lys174, Ala162 and Pro129 of the R Domain. Gln 19 hydrogen bonds multiple times with the oxygen atoms of Ser176 and also with the oxygen atom on Tyr88. Electrostatic interactions are seen between Glu35 and Lys174 where the carboxyl group of Glu35 forms an ionic bond with the ammonia group of the Lys174 residue. The sum total of interactions within the cleft between the two domains ensure that the lobes do not move with respect to one another. The active site contains a catalytic diad made up of Cysteine-25 and Histidine-159. Aspartate-158 also plays a role in catalysis but it is not considered part of the diad. Papain's active site can accommodate seven amino acids of a substrate. When the peptide is cleaved, the first four resides reside on the amino side of the peptide bond while the other three reside on the carboxyl side. Papain prefers to cleave at: (hydrophobic)-(Arg or Lys)- cleaves here -(not Val). Hydrophobic is Ala, Val, Leu, Ile, Phe, Trp, or Tyr. spinqualitylabelsall models carboxymethylated papain complexed with human Stefin B Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Catalytic Inhibitors Stefin B acts as a competitive inhibitor to cysteine proteases-- it binds tightly but reversibly to the papain active site. Stefin inhibitors are characterized by Mr of about 11,000, no disulfide bonds and no associated carbohydrates. In Stefin B, the Gly9 residue along with two hairpin loops form a "wedge" complementary to the active site groove of papain. This wedge makes extensive and tight interactions with papain and a total of 128 intermolecular atom-atom interactions occur. Residue segments Met6-Pro11, Gln53-Asn59, Gln101-His104 and Tyr124-Phe125 on the wedge all have some interaction to the enzyme though not always direct. All residues from the base and both sides of the active site cleft are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159). There are a small number of direct hydrogen bonds between stefin B and papain, however there are many more polar interactions mediated by solvent bridges. Thirteen solvent molecules bridge polar residues of the enzyme and inhibitor. Seventeen hydrogen bonds are made with a solvent molecule and stefin. Fourteen of these bridges form a papain contact. The rest of the interactions are largely hydrophobic-- involving apolar Van der Waals interactions. References