Papain
Papin is a member of the thiol protease family, which is a large, well-studied group with a well-known "catalytic triad" motif in which a cysteine acts as the catalytic nucleophile, activated by His and Asn. Papin catalyses the hydrolysis of proteins with broad specificity for peptide bonds, but shows a marked preference for an amino acid bearing a large hydrophobic side chain at the P2 position and does not accept Val in P1'.
Reference Protein and Structure
- Sequence
-
P00784
(3.4.22.2)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Carica papaya (Papaya)
- PDB
-
9pap
- STRUCTURE OF PAPAIN REFINED AT 1.65 ANGSTROMS RESOLUTION
(1.65 Å)
- Catalytic CATH Domains
-
3.90.70.10
(see all for 9pap)
Enzyme Reaction (EC:3.4.22.2)
+
→
+
Alternative enzyme names: Papaya peptidase I, Papaine, Papayotin, Summetrin, Velardon,
Enzyme Mechanism
Introduction
The reaction is a hydrolysis of a peptide, ester, thio-ester or thiono-ester bond, via a covalent intermediate, resulting from nucleophilic attack of the active-site thiol group on the carbonyl carbon of the amide or ester scissile bond.
Catalytic Residues Roles
| UniProt | PDB* (9pap) | ||
| Asn308 | Asn175A | Activates the histidine of the catalytic Cys-His-Asn triad. | activator, hydrogen bond acceptor, electrostatic stabiliser |
| Cys158 | Cys25A | Acts as a catalytic nucleophile, becomes covalently attached to the protein substrate during the course of the reaction. | covalently attached, hydrogen bond donor, nucleophile, nucleofuge, proton acceptor, electrostatic stabiliser |
| His292 | His159A | Acts as a general acid/base. Also stabilises the catalytic cysteine in the thiolate form. | hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser |
| Gln152, Cys158 (main-N) | Gln19A, Cys25A (main-N) | Forms the oxyanion hole that stabilises the intermediates and transition states formed during the course of the reaction. | hydrogen bond donor, electrostatic stabiliser |
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file;
y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain;
C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.
Chemical Components
bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation, proton transfer, unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, overall product formed, bimolecular nucleophilic substitution, intermediate terminated, native state of enzyme regeneratedReferences
- Theodorou LG et al. (2001), Biochemistry, 40, 3996-4004. Insight into the Catalysis of Hydrolysis of Four Newly Synthesized Substrates by Papain: A Proton Inventory Study. DOI:10.1021/bi001615b. PMID:11300780.
- Fekete A et al. (2016), Phys Chem Chem Phys, 18, 32847-32861. Modeling the archetype cysteine protease reaction using dispersion corrected density functional methods in ONIOM-type hybrid QM/MM calculations; the proteolytic reaction of papain. DOI:10.1039/c6cp06869c. PMID:27883128.
- Wei D et al. (2013), Biochemistry, 52, 5145-5154. Reaction pathway and free energy profile for papain-catalyzed hydrolysis of N-acetyl-Phe-Gly 4-nitroanilide. DOI:10.1021/bi400629r. PMID:23862626.
- Harrison MJ et al. (1997), J Am Chem Soc, 119, 12285-12291. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. DOI:10.1021/ja9711472.
- Johnston SC et al. (1997), EMBO J, 16, 3787-3796. Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 Å resolution. DOI:10.1093/emboj/16.13.3787. PMID:9233788.
- Pedersen LC et al. (1994), Protein Sci, 3, 1131-1135. Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules. DOI:10.1002/pro.5560030720. PMID:7920263.
- Storer AC et al. (1994), Methods Enzymol, 244, 486-500. [33] Catalytic mechanism in papain family of cysteine peptidases. DOI:10.1016/0076-6879(94)44035-2. PMID:7845227.
Step 1. The thiolate of Cys25 attacks the carbonyl carbon of the peptide bond in a nucleophilic addition.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gln19A | hydrogen bond donor, electrostatic stabiliser |
| Cys25A | hydrogen bond donor, electrostatic stabiliser |
| His159A | hydrogen bond donor, electrostatic stabiliser |
| Asn175A | hydrogen bond acceptor, electrostatic stabiliser |
| Cys25A (main-N) | electrostatic stabiliser |
| Cys25A | nucleophile |
Chemical Components
ingold: bimolecular nucleophilic addition, overall reactant used, enzyme-substrate complex formation, intermediate formation
Step 2. The oxyanion initiates an elimination that cleaves the peptide bond, the released N-terminus of the protein deprotonates from His159.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gln19A | hydrogen bond donor, electrostatic stabiliser |
| Cys25A | covalently attached, hydrogen bond donor, electrostatic stabiliser |
| His159A | hydrogen bond donor |
| Asn175A | hydrogen bond acceptor, electrostatic stabiliser |
| Cys25A (main-N) | electrostatic stabiliser |
| His159A | proton donor |
Chemical Components
proton transfer, ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, intermediate collapse, intermediate formation, overall product formed
Step 3. His159 deprotonates a water molecule, which attacks the carbonyl carbon of the cysteine bound intermediate in a nucleophilic substitution, resulting in the C-terminal product and the regeneration of the ground state of Cys25.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gln19A | hydrogen bond donor, electrostatic stabiliser |
| Cys25A | hydrogen bond donor, electrostatic stabiliser |
| His159A | hydrogen bond donor, hydrogen bond acceptor |
| Asn175A | hydrogen bond acceptor, activator |
| Cys25A (main-N) | electrostatic stabiliser |
| Cys25A | nucleofuge |
| His159A | proton acceptor |
Chemical Components
proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, enzyme-substrate complex cleavage, intermediate collapse, intermediate terminated, overall product formed
Step 4. Cys 25 accepts a proton from His 159 regenerating the native state of the active site.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Gln19A | electrostatic stabiliser |
| Cys25A (main-N) | electrostatic stabiliser |
| Asn175A | electrostatic stabiliser |
| His159A | proton donor |
| Cys25A | proton acceptor |
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