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Papain

Introduction

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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

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 Contents 1 Structural Elements 2 Active Site 3 Distribution of Residues 4 Ligand interactions and Pseudo Substrates Structural Elements 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 consists of 17 strands and 7 giving it a composition 21% and 25% respectively. [7] 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 , serve to hold papain's tertiary structure together. [8] Contacts between Papain Subunits.[9]. Papain's two subunits are held together with "arm" linkage where one end of the protein chain holds the opposite domain. In papain's case the "arm crossing" primarily occurs on or near the surface. [9] Active Site Located in the cleft between its domains, the active site consists of seven subsites (S1-S4 and S1’-S3’) each accommodating one amino acid residue of a substrate (P1-P4 and P1’-P3’). [10] The specificity of the active site is controlled by the S2 subsite which is a hydrophobic pocket that accommodates the P2 side chain of the substrate. Particularly at this subsite, papain shows specific substrate preferences for bulky hydrophobic or aromatic residues. On the other hand, outside of the S2 subsite preferences, the active site appears to exhibit a lack of clearly defined residue selectivity from within. [11] The primarily consist of three main residues Cys 25, His 159, and Asn 175 holding resemblance to the catalytic triad of chymotrypsin [12][13]. 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-! It is postulated that "a two-state mechanism" takes place instead of a "single steric mechanism." [12] 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. [14] 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. [8] 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. [15] 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. [8] Ligand interactions and Pseudo Substrates Papain is said to have 29 identifiable methanol molecules that encircle around it as . The polarity of the ligands result in hydrogen bonding interactions, possibly providing further stability for papain structure. [8] 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 . 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. [16] Through modeling, more favorable energetics has also been revealed when hydrophobic and aromatic parts of the ligand occupy the S2, S3, and S1' subsites with at least three hydrogen bonding contacts between the protein conserved binding site residues and the ligand. [17]

Catalytic Mechanism

General mechanism of papain catalysis^[18].

Papain's catalytic mechanism is like serine proteases. Its catalytic triad of residues Cys 25- His159- Asn-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. ^[18]

Other Interaction with Inhibitors and Effectors

Except for valine, papain prefers to cleave at hydrophobic residues alanine, leucine, isoleucine, phenylalanine, tryptophan, or tyrosine ^[20]. In light of the describe catalytic mechanism it makes sense that substances such as cysteine, sulfide/sulfite, heavy metal chelating agents like EDTA, and N-bromosuccinimide act as activators of the enzyme while PMSF, Hg2+ and other heavy metals, cystatin, leupeptin, sulfhydryl binding agents, carbonyl reagents, and alkylating agents serve as inhibitors. ^[21][22]

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! ^[23]

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} [3] Jane S. Richardson
10. ↑ Schechter and Berger 1967
11. ↑ Kimmel and Smith 1954
12. ↑ ^{12.0 12.1} 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
13. ↑ 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
14. ↑ [4] The Journal of Biological Chemistry
15. ↑ [5] WebMD
16. ↑ 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
17. ↑ Skern T, Fita I, Guarne A. A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase. J Gen Virol. 1998 Feb;79 ( Pt 2):301-7. PMID:9472614
18. ↑ ^{18.0 18.1} [6] University of Maine
19. ↑ [7] Worthington Biochemical Corporation
20. ↑ [8] Sigma Aldrich Papain
21. ↑ Nicklin MJ, Barrett AJ. Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin. Biochem J. 1984 Oct 1;223(1):245-53. PMID:6388564 doi:10.1042/bj2230245
22. ↑ [9] Biozym
23. ↑ 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