From Proteopedia

Revision as of 03:49, 14 November 2011

Papain

Introduction

Cartoon Peak at Pepsin

DID YOU KNOW?

. 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][3]

Papain. Lights. Camera. Action!

Structure

Papain's polypeptide chain consists of 212 amino acid residues which fold to form a groove containing the active site between its two domains. Its consists of 17 strands and 7 giving it a composition 21% and 25% respectively. ^[4] 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, for example , serve to hold papain's tertiary structure together. ^[5]

The primarily consist of three main residues Cys25-His159-Asn175 that resembles the catalytic triad of chymotrypsin ^[6][7]. However growing studies are showing that the mechanism behind catalysis may actually involve a double catalytic site - consisting of Cys25-His159-Asn175 and Cys25-His159- ! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." ^[8] 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 the Asn 175 plays than originally thought. It should be noted however, that alteration to the 175 side chain resulted in less thermal stability lending thought to Asn 175 playing a more structural conservative role rather than catalytic. ^[9]

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. ^[10]

Distribution of Residues

Although Papain has a scattered distribution of , it can be seen to have more basic residues than acidic, shedding understanding into the application of its use as a digestive supplement. ^[11] Seeing its further shows remaining mostly on the exterior while 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. ^[12]

Ligands interactions and Pseudo Substrates

Papain is said to have 29 methanol molecules that encircle around it as . The polarity of the ligands result in hydrogen bonding interactions, possibly providing further stability for papain structure. ^[13]

is part of a series known as CLIK inhibitors and was used on Papain for assessment of specificity in inhibition. The difference in structure between Papain-CLIK 148 complex and orginial papain is not very drastic. The changes result primarily from alterations in surface proteins except where a covalent bond is formed between the C2 on . The primary 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 . Hydrogen bonding is observed between the oxygens in . 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. ^[14]

Catalytic Mechanism

General mechanism of papain catalysis^[15].

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 resultant covalent, tetrahedral intermediate. Subsequently, nitrogen in the peptide bond in 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 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 and later renegerates the enzyme. ^[16]

Fun Trivia

Remember the 2002 SARS (Severe Acute Respiratory Syndrome) epidemic that placed global health 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. ^[17]

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. ↑ [2]9PAP PDB
5. ↑ 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
6. ↑ 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
7. ↑ 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
8. ↑ 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
9. ↑ [3] The Journal of Biological Chemistry
10. ↑ 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
11. ↑ [4] WebMD
12. ↑ 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
13. ↑ 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
14. ↑ 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
15. ↑ [5] University of Maine
16. ↑ [6] University of Maine
17. ↑ 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