Sandbox 35

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. Papain Introduction Cartoon Peak at Pepsin DID YOU KNOW? Papain. Meat tenderizer. Old time home remedy for insect, jellyfish, and stingray stings. 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? 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. Papain. Lights. Camera. Action! spinqualitylabelsall models Structure of HMG-CoA reductase (PDB entry 9pap) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structure 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 secondary structure consists of 17 beta sheet strands and 7 alpha helices giving it a composition 21% and 25% respectively. 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 Cys 22-Cys 63, serve to hold papain's tertiary structure together. The active site primarily consist of three main residues Cys 25, His 159, and Asn 175 holding resemblance to the catalytic triad of chymotrypsin . 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- Asp 158! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." In addition, replacement of Asn 175 with other residues such as Ala mutants, reveals a decrease in kcat (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. 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. Distribution of Residues Although papain has a scattered distribution of acidic and basic residues, it can be seen to have more basic residues than acidic, shedding understanding into the application of its use as a digestive supplement. Seeing its polar and non-polar residues further shows polar residues remaining mostly on the exterior while non-polar residues 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. Ligand interactions and Pseudo Substrates Papain is said to have 29 identifiable methanol molecules that encircle around it as ligands. The polarity of the ligands result in hydrogen bonding interactions, possibly providing further stability for papain structure. Cathepsin L specific inhibitor 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 CLIK 148 and Cys 25 residue. The primary interactions 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 Trp 177 and CLIK 148. Hydrogen bonding is observed between the oxygens in CLIK 148 to Gln 19 and Gly 66 residues. 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. Catalytic Mechanism General mechanism of papain catalysis. Papain's catalytic mechanism is like serine proteases. Its catalytic triad of residues Cys 25- His159- Arg-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. Fun Trivia 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! References