Papain: Difference between revisions

The seven subsites of Papain have various preferences for substrate residues. Through a variety of experiments, Berger & Schechter<ref>PMID:4399049</ref>, were able to show that S1 binds alanine better than glycine, and the larger side chains of lysine, arginine, leucine, and phenylalanine better than alanine. Thus showing that binding in S1 is predominantly hydrophobic. S2 prefers a phenylalanine or a valine residue. Interestingly enough, S2 binds to hydrophobic residues of both short and long peptide chains. They were able to show that subsites S1' and S2' are strongly stereospecific. The conclusion of their research was that Papain's binding site residues show a strong stereospecificity, special interactions, and space limitations.<ref>PMID:4399049</ref>

It was once thought that cysteine proteases, like serine proteases, contained a <scene name='Papain/Catalytic_triad/1'>catalytic triad</scene>, consisting of Cys-25, His-159, and Arg-175 <ref> PMID:8140097</ref>.  However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis.  Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.<ref name="Shokhen">[http://www.ncbi.nlm.nih.gov/pubmed/19688822]Shokhen M, N Khazanov, and A Albeck. 2009. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain. Proteins. 77(4):916-26.</ref><ref name="Noble">[http://www.ncbi.nlm.nih.gov/pubmed/11042128]Noble MA, Gul S, Verma CS, Brocklehurst K. 2000. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry. Biochem J. 2000 351: 723-33.</ref> Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.<ref name="U Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases.  The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an <scene name='Papain/Oxyanion_hole/1'>oxyanion hole</scene>, formed in large part by Gln-19. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme.  This entire mechanism is shown below<ref name="Harrison">[http://pubs.acs.org/doi/abs/10.1021/ja9711472] Harrison, M.J., N.A. Burton, and I.H. Hillier. 1997. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. J. Am. Chem. Soc. 119: 12285-12291</ref>.:

[[Image:Papainmech6.jpg|400px|center|thumb| General mechanism of papain catalysis <ref name="U Maine" />.  Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.]]

==='''Leupeptin'''===

</StructureSection>

==Common Uses==

[[Image:RX2.jpg|200px|right|thumb|<ref>[http://www.123rf.com/photo_10020414_medical-symbol-rx.html] RX </ref>]]

A recent New York Times article featured papain and other digestive enzymes. With the number of individuals suffering from irritable bowel syndrome and other gastrointestinal issues, many people are turning toward natural digestive aid supplements like papain.  The author even talks about the use of papain along with a pineapple enzyme, bromelain, in cosmetic facial masks.  Dr. Adam R. Kolker (a plastic surgeon) is quoted in the article saying that "For skin that is sensitive, enzymes are wonderful."  He bases these claims off the idea that proteases like papain help to break peptide bonds holding dead skin cells to the live skin cells.<ref> [http://www.nytimes.com/2012/02/23/fashion/enzymes-once-sidelined-try-to-grab-the-spotlight.html] Enzymes Try to Grab the Spotlight </ref>

===Commercial and Biomedical===

Papain digests most proteins, often more extensively than pancreatic proteases. It has a very broad specificity and is known to cleave peptide bonds of basic amino acids and leucine and glycine residues, but prefers amino acids with large hydrophobic side chains. This non-specific nature of papain's hydrolase activity has led to its use in many and varied commercial products.  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 and marketed as a digestive supplement.  <ref name="Web MD">  Finally, papain has several common uses in general biomedical research, including a gentle cell isolation agent, production of glycopeptides from purified proteoglycans, and solubilization of integral membrane proteins.  It is also notable for its ability to specifically cleave IgG and IgM antibodies above and below the disulfide bonds that join the heavy chains and that is found between the light chain and heavy chain. This generates two monovalent Fab segments, that each have a single antibody binding sites, and an intact Fc fragment.<ref name="Worthington" />  

==Fun Trivia==

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 papain's five alpha-helices and seven beta-sheets, 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>   

==References==  

<ref group="xtra">PMID:8140097</ref>

==3D Structures of Papain==

[[3e1z]] - ''Trypanosoma cruzi''

[[Category:Topic Page]]

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