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Located in the cleft between its domains, the <scene name='Sandbox_35/Active_site_papain/4'>active site</scene> primarily consist of three main residues Cys 25, His 159, and Asn 175 holding resemblance to the catalytic triad of chymotrypsin <ref name="Wang">PMID: 8140097</ref><ref>PMID: 2397208</ref>. 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-
Located in the cleft between its domains, the <scene name='Sandbox_35/Active_site_papain/4'>active site</scene> primarily consist of three main residues Cys 25, His 159, and Asn 175 holding resemblance to the catalytic triad of chymotrypsin <ref name="Wang">PMID: 8140097</ref><ref>PMID: 2397208</ref>. 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-
<scene name='Sandbox_35/Active_site_papain/5'>Asp 158</scene>! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." <ref name="Wang" /> In addition, replacement of Asn 175 with other residues such as Ala mutants, reveals a decrease in kcat revealing 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. <ref>[http://www.jbc.org/content/270/28/16645.abstract] The Journal of Biological Chemistry </ref>
<scene name='Sandbox_35/Active_site_papain/5'>Asp 158</scene>! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." <ref name="Wang" /> In addition, replacement of Asn 175 with other residues such as Ala mutants, reveals a decrease in kcat revealing 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. <ref>[http://www.jbc.org/content/270/28/16645.abstract] The Journal of Biological Chemistry </ref>
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Papain's two domains are held together
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. <ref name="Kamphuis" />
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. <ref name="Kamphuis" />

Revision as of 17:02, 8 March 2012

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.


Contents

Papain

Introduction

Did you know?

Cartoon Peak at Papain
Cartoon Peak at Papain

. Meat tenderizer. Old time home remedy for insect, jellyfish, and stingray stings[1]. 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. [2] The protein is primarily secreted with its pro-region enabling transport from zymogen to lysosome through membrane association and mediation. [3]

Historicity

Papain made its first appearance in the Calcutta Medical Journal entitled “The Solvent Action of Papaya Juice on Nitrogenous Articles of Food” when G.C Roy was investigating the enzyme in 1873. In the late 19th century, Wurtz and Bouchut dubbed the partially purified enzyme "papain." [4] At the time, it was viewed as proteolytically active constituent in the latex of tropical papaya fruit. [5] As separation and purification techniques improved, pure papain was able to be isolated. In becoming the second enzyme to attain an X-ray crystallized structure and the first cysteine protease to behold an identifiable structure, papain fueled greater advances in enzymatic studies. [6]


Papain. Lights. Camera. Action!

Structure of HMG-CoA reductase (PDB entry 9pap)

Drag the structure with the mouse to rotate

Catalytic Mechanism

General mechanism of papain catalysis.
General mechanism of papain catalysis[14].


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. [14]

The specificity of Papain
Image:Papain Simple Cleavage.jpg
Simple Overview of Papain Cleavage


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! [15]


References

  1. ↑ [1] Ameridan International
  2. ↑ Rawlings ND, Barrett AJ. Families of cysteine peptidases. Methods Enzymol. 1994;244:461-86. PMID:7845226
  3. ↑ Yamamoto Y, Kurata M, Watabe S, Murakami R, Takahashi SY. Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases. Curr Protein Pept Sci. 2002 Apr;3(2):231-8. PMID:12188906
  4. ↑ Menard and Storer 1998
  5. ↑ Wurtz and Bouchut 1879
  6. ↑ Drenth J, Jansonius JN, Koekoek R, Swen HM, Wolthers BG. Structure of papain. Nature. 1968 Jun 8;218(5145):929-32. PMID:5681232
  7. ↑ [2]9PAP PDB
  8. ↑ 8.0 8.1 8.2 8.3 Kamphuis IG, Kalk KH, Swarte MB, Drenth J. Structure of papain refined at 1.65 A resolution. J Mol Biol. 1984 Oct 25;179(2):233-56. PMID:6502713
  9. ↑ 9.0 9.1 Wang J, Xiang YF, Lim C. The double catalytic triad, Cys25-His159-Asp158 and Cys25-His159-Asn175, in papain catalysis: role of Asp158 and Asn175. Protein Eng. 1994 Jan;7(1):75-82. PMID:8140097
  10. ↑ Ménard R, Khouri HE, Plouffe C, Dupras R, Ripoll D, Vernet T, Tessier DC, Lalberté F, Thomas DY, Storer AC. A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain. Biochemistry. 1990 Jul 17;29(28):6706-13. PMID:2397208 doi:10.1021/bi00480a021
  11. ↑ [3] The Journal of Biological Chemistry
  12. ↑ [4] WebMD
  13. ↑ Tsuge H, Nishimura T, Tada Y, Asao T, Turk D, Turk V, Katunuma N. Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex. Biochem Biophys Res Commun. 1999 Dec 20;266(2):411-6. PMID:10600517 doi:10.1006/bbrc.1999.1830
  14. ↑ 14.0 14.1 [5] University of Maine
  15. ↑ Barretto N, Jukneliene D, Ratia K, Chen Z, Mesecar AD, Baker SC. The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity. J Virol. 2005 Dec;79(24):15189-98. PMID:16306590 doi:10.1128/JVI.79.24.15189-15198.2005
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