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Papain

Introduction

Papain is a cysteine protease, also known as papaya proteinase I, from the peptidase C1 family (E.C. 3.4.22.2).^[1] It functions as an endopeptidase, amidase, and esterase.^[2] Its optimal activity values for pH lie between 6.0 and 7.0, and its optimal temperature for activity is 65 °C. Its pI values are 8.75 and 9.55. Papain is best visualized at a wavelength of 278 nm. ^[3] While only consisting of a single peptide chain, papain has two domains that form a cleft in which the active site lies.^[4] Naturally found in the latex of the papaya fruit, one of the most common uses of papain is as a meat tenderizer because of its ability to hydrolyze esters and amides.^[5] Another common use is as a digestive aid. Papaya is commonly referenced as a preferred fruit for those suffering from gastroesophageal reflux disease due to its ability to help the the stomach with digestion of complex proteins.

History

Papain's enzymatic use was first discovered in 1873 by G.C. Roy who published his results in the Calcutta Medical Journal in the article, "The Solvent Action of Papaya Juice on Nitrogenous Articles of Food."^[2] In 1879, papain was named officially by Wurtz and Bouchut, who managed to partially purify the product from the sap of papaya.^[2]. It wasn't until the mid-twentieth century that the complete purification and isolation of papain was achieved. In 1968, Drenth et al. determined the structure of papain by x-ray crystallography, making it the second enzyme whose structure was successfully determined by x-ray crystallography. Additionally, papain was the first cysteine protease to have its structure identified. ^[2] In 1984, Kamphuis et al. determined the geometry of the active site, and the three-dimensional structure was visualized to a 1.65 Angstrom solution.^[6] Today, studies continue on the stability of papain, involving changes in environmental conditions, in addition to testing of inhibitors such as phenylmethanesulfonylfluoride (PMSF), TLCK, TPCK, aplh2-macroglobulin, heavy metals, AEBSF, antipain, cystatin, E-64, leupeptin, sulfhydryl binding agents, carbonyl reagents, and alkylating agents.^[2]

Structure

Interestingly enough, in The Structure of Papain Refined at 1.65 A Resoltion, Kamphuis et al. discovered interesting information on between molecules of papain in solution. These contacts, communicated in Table 7 of their paper, consist of nine hydrogen-bond connections and three ionic interactions. The strongest salt bridge exists between .^[6]

Solvent Interactions

Papain binds both via hydrogen bonds. This solvent mixture of 62%, (w/w) in was used in order to obtain a C-type crystal, which is . In addition to the methanol molecules, papain also has an interesting hydrogen bonding with water molecules. A refined crystal structure of papain, involving a restrained least-squares procedure at 1.65 A, with an estimated accuracy of 0.1 A, revealed that water forms something similar to a hydration shell around individual molecules of papain. The interaction of papain with these water molecules leads to less interaction between papain molecules, and contributes to the stability of the crystal structure of papain.^[6]

A general mechanism of papain catalysis^[8].

Specificity

Papain digests a large variety of proteins, with a very broad specificity. Its consists of the residues cysteine-25, histidine-159, and asparagine-175. While asparagine-175 is included with the active site, it does not play a direct role in the mechanism of papain. Rather, it supports the It cleaves the peptide bonds of basic amino acids, leucine and glycine by nucleophilic attack with its sulfhydryl group on cysteine-25 ^[9]. It also hydrolyzes esters and amides. It prefers amino acids that bear large hydrophobic side chains at the P2 position, and will not accept valine at the P1' position. ^[1]

Catalytic Mechanism

The mechanism of cysteine proteases is very similar to that of serine proteases. However, instead of requiring a triad, papain only requires a diad. The sulfhydryl group on cysteine executes a nucleophilic attack on the peptide bond of the protein it wishes to cleave. Asparagine-175 keeps histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual mechanism. Opening up the carbonyl, the sulfhydryl group of CYS-25 is stabilized by HIS-159. As the carbonyl reforms, the peptide bond is broken, leaving the amide group to fend for itself.^[10]

Inhibitors

There are many inhibitors for papain because of its broad specificity. It is often used as a model enzyme for those in the papain superfamily, such as cathepsin L and cathepsin K. The inhibition of papain is usually due to active site restriction of cysteine-25 and histidine-159. An interesting inhibitor for cathepsin L developed using papain as the model protease is that of . This inhibitor uses both prime and non-prime sites to inhibit papain.^[11] In two different cathepsin K inhibitors, referenced PDB codes and , it is evident that the inhibitor binds with much closer proximity than that of Clik148.^[12]

    Other inhibitors, such as , illustrated in magenta, are much more complex in their . The human stefin B molecule has a five stranded beta-sheet that wraps around a five turn alpha-helix. The interface between human stefin B and papain is very tightly packed with 16% of stefin B becoming embedded within papain. A total of 128 

<4 A occur within the cleft in papain. Through this study, Stubbs et al. were able to conclude that cysteine proteinase inhibitors are "fundamentally different to [those] observed for serine proteinase inhibitors."^[13]

References

1. ↑ ^{1.0 1.1} http://www.uniprot.org/uniprot/P00784
2. ↑ ^{2.0 2.1 2.2 2.3 2.4} http://www.worthington-biochem.com/pap/default.html
3. ↑ http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html
4. ↑ ^{4.0 4.1} http://www.ebi.ac.uk/thornton-srv/databases/cgi-bin/pdbsum/GetPage.pl?pdbcode=9pap&template=clefts.html&r=speedfill
5. ↑ IUBMB Enzyme Nomenclature: www.chem.qmul.ac.uk/iubmb/enzyme/EC3/4/22/2.html
6. ↑ ^{6.0 6.1 6.2} 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
7. ↑ http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html
8. ↑ [1] University of Maine
9. ↑ http://www.ebi.ac.uk/QuickGO/GTerm?id=GO:0004197
10. ↑ [2] University of Maine
11. ↑ 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
12. ↑ LaLonde JM, Zhao B, Smith WW, Janson CA, DesJarlais RL, Tomaszek TA, Carr TJ, Thompson SK, Oh HJ, Yamashita DS, Veber DF, Abdel-Meguid SS. Use of papain as a model for the structure-based design of cathepsin K inhibitors: crystal structures of two papain-inhibitor complexes demonstrate binding to S'-subsites. J Med Chem. 1998 Nov 5;41(23):4567-76. PMID:9804696 doi:10.1021/jm980249f
13. ↑ Stubbs MT, Laber B, Bode W, Huber R, Jerala R, Lenarcic B, Turk V. The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction. EMBO J. 1990 Jun;9(6):1939-47. PMID:2347312

External References

Wikipedia