Papain: Difference between revisions

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<scene name='Papain/Substrate_binding_sites/1'>Substrate binding sites.</scene><ref>PMID:4399049</ref>

===Catalytic Mechanism===

It was once thought that cysteine proteases, like serine proteases, contained a <scene name='Sandbox_30/Papain_ligand_active-site/3'>catalytic triad</scene>, consisting of Cys-25, His-159, and Arg-175. However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis. Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.<ref name="Shokhen">[http://www.ncbi.nlm.nih.gov/pubmed/19688822]Shokhen M, N Khazanov, and A Albeck. 2009. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain. Proteins. 77(4):916-26.</ref><ref name="Noble">[http://www.ncbi.nlm.nih.gov/pubmed/11042128]Noble MA, Gul S, Verma CS, Brocklehurst K. 2000. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry. Biochem J. 2000 351: 723-33.</ref> Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.<ref name="U Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases. The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an oxyanion hole, formed in large part by <scene name='Papain/1pop_sam_leupeptin_cat_gln19/1'>Gln-19</scene>. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme. This entire mechanism is shown below:

It was once thought that cysteine proteases, like serine proteases, contained a <scene name='Sandbox_30/Papain_ligand_active-site/3'>catalytic triad</scene>, consisting of Cys-25, His-159, and Arg-175. However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis. Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.<ref name="Shokhen">[http://www.ncbi.nlm.nih.gov/pubmed/19688822]Shokhen M, N Khazanov, and A Albeck. 2009. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain. Proteins. 77(4):916-26.</ref><ref name="Noble">[http://www.ncbi.nlm.nih.gov/pubmed/11042128]Noble MA, Gul S, Verma CS, Brocklehurst K. 2000. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry. Biochem J. 2000 351: 723-33.</ref> Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.<ref name="U Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases. The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an oxyanion hole, formed in large part by <scene name='Papain/1pop_sam_leupeptin_cat_gln19/1'>Gln-19</scene>. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme. This entire mechanism is shown below<ref name="Harrison">[http://pubs.acs.org/doi/abs/10.1021/ja9711472] Harrison, M.J., N.A. Burton, and I.H. Hillier. 1997. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. J. Am. Chem. Soc. 119: 12285-12291</ref>.:

[[Image:Papainmech6.jpg|400px|center|thumb| General mechanism of papain catalysis <ref name="U Maine" />. Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.]]

<ref name="Harrison">[http://pubs.acs.org/doi/abs/10.1021/ja9711472] Harrison, M.J., N.A. Burton, and I.H. Hillier. 1997. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. J. Am. Chem. Soc. 119: 12285-12291</ref>.

</StructureSection>

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@@ Line 23: / Line 23: @@
 <scene name='Papain/Substrate_binding_sites/1'>Substrate binding sites.</scene><ref>PMID:4399049</ref>
 ===Catalytic Mechanism===
-It was once thought that cysteine proteases, like serine proteases, contained a <scene name='Sandbox_30/Papain_ligand_active-site/3'>catalytic triad</scene>, consisting of Cys-25, His-159, and Arg-175.  However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis.  Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.<ref name="Shokhen">[http://www.ncbi.nlm.nih.gov/pubmed/19688822]Shokhen M, N Khazanov, and A Albeck. 2009. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain. Proteins. 77(4):916-26.</ref><ref name="Noble">[http://www.ncbi.nlm.nih.gov/pubmed/11042128]Noble MA, Gul S, Verma CS, Brocklehurst K. 2000. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry. Biochem J. 2000 351: 723-33.</ref> Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.<ref name="U Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases.  The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an oxyanion hole, formed in large part by <scene name='Papain/1pop_sam_leupeptin_cat_gln19/1'>Gln-19</scene>. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme.  This entire mechanism is shown below:
+It was once thought that cysteine proteases, like serine proteases, contained a <scene name='Sandbox_30/Papain_ligand_active-site/3'>catalytic triad</scene>, consisting of Cys-25, His-159, and Arg-175.  However, site-directed mutagenesis-based studies have demonstrated that Arg-175 is not directly involved in catalysis.  Although Arg-175 is clearly important for the enzyme's activity (an Arg175Ala mutation reduces its activity to undetectable levels), this residue neither reacts with the substrate nor modulates the pKa of reacting residues, and therefore cannot be considered catalytic.<ref name="Shokhen">[http://www.ncbi.nlm.nih.gov/pubmed/19688822]Shokhen M, N Khazanov, and A Albeck. 2009. Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain. Proteins. 77(4):916-26.</ref><ref name="Noble">[http://www.ncbi.nlm.nih.gov/pubmed/11042128]Noble MA, Gul S, Verma CS, Brocklehurst K. 2000. Ionization characteristics and chemical influences of aspartic acid residue 158 of papain and caricain determined by structure-related kinetic and computational techniques: multiple electrostatic modulators of active-centre chemistry. Biochem J. 2000 351: 723-33.</ref> Arg-175 is believed to keep histidine-159 in its stabilized imidazole form, while both histidine-159 and cysteine-25 take part in the actual catalytic mechanism.<ref name="U Maine">[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref> Despite this, the basic mechanism of papain-catalyzed proteolysis proceeds much like that of serine proteases.  The mechanism begins when a peptide binds to the active site. Cys-25 is then deprotonated by His-159 and attacks the substrate carbonyl carbon. This forms a covalent, tetrahedral intermediate that is stabilized by an oxyanion hole, formed in large part by <scene name='Papain/1pop_sam_leupeptin_cat_gln19/1'>Gln-19</scene>. Next, His-159 acts as a general acid, protonating the nitrogen in the peptide bond, which acts as a leaving group as the carbonyl reforms. This now free C-terminal portion of the peptide is released. Water then enters the active site and attacks the carbonyl carbon while it is deprotonated by His-159, again forming an oxyanion hole-stabilized tetradral covalent intermediate. Finally, the carbonyl reforms and the Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme.  This entire mechanism is shown below<ref name="Harrison">[http://pubs.acs.org/doi/abs/10.1021/ja9711472] Harrison, M.J., N.A. Burton, and I.H. Hillier. 1997. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. J. Am. Chem. Soc. 119: 12285-12291</ref>.:
 [[Image:Papainmech6.jpg|400px|center|thumb| General mechanism of papain catalysis <ref name="U Maine" />.  Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.]]
-<ref name="Harrison">[http://pubs.acs.org/doi/abs/10.1021/ja9711472] Harrison, M.J., N.A. Burton, and I.H. Hillier. 1997. Catalytic Mechanism of the Enzyme Papain: Predictions with a Hybrid Quantum Mechanical/Molecular Mechanical Potential. J. Am. Chem. Soc. 119: 12285-12291</ref>.
 </StructureSection>