Papain: Difference between revisions

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Papain's secondary structure is composed of 21% <scene name='Papain/Ke_betasheets/1'>beta sheets</scene> (45 residues comprising 17 sheets) and 25% <scene name='Papain/Ke_alphahelices/1'>alpha helices</scene> (51 residues comprising 7 helices). The rest of the residues, accounting for over 50% of the enzymes structure, make up ordered non-repetative sequences.<ref name="RSCB PDB">http://www.rcsb.org/pdb/explore/explore.do?structureId=9PAP</ref> These secondary structures may be traced from the N- to C-terminus by means of <scene name='Papain/Lm_elemental/1'>differential coloration</scene>. As shown in this scene, the red end begins the protein at the N-terminus, and can be traced through the colors of the rainbow to the blue end at the C-terminus. These secondary structures form as a result of favorable hydrogen bonding interactions within the polypeptide backbone. Meanwhile, secondary structures are kept in place by hydrophobic interactions and hydrogen bonds between sidechains of adjacent structures. For example, the <scene name='Papain/Papain_sam_centralhelix/1'>first helix</scene> (residues 25-42) is maintained as a result of <scene name='Papain/Papain_sb_helix1_hbonds/1'>hydrogen bonds</scene> between backbone carbonyl atoms and the hydrogen on the amide nitrogen four residues away. However, <scene name='Papain/Papain_sam_nohbondswhelix1/1'>no hydrogen bonds</scene> are present between this helix and the rest of the protein, suggesting that this helix is coordinated primarily by hydrophobic interactions. This is reasonable given its central location in the enzyme. As expected, the helix contains many <scene name='Papain/Papain_sb_helix1_hydrophobic/1'>hydrophobic residues</scene> (red residues are hydrophilic).

<scene name='Papain/Ke_salt_bridges/1'>Salt bridges</scene> strongly contribute to the tertiary structure of papain.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue. The tertiary structure of papain is also maintained by three <scene name='Papain/9pap_sam_disulfides/1'>disulfide bonds</scene>, which connect <scene name='Papain/9pap_sam_disulfides_22-63/1'>Cys-22 to Cys63</scene>, <scene name='Papain/9pap_sam_disulfides_56-95/1'>Cys-56 to Cys-95</scene>, and <scene name='Papain/9pap_sam_disulfides_153-200/1'>Cys-153 to Cys-200</scene><ref name="9PAP PDB" />. These disulfide bonds are likely important in conserving the structural integrity of the enzyme as it operates in extracellular environments at high temperatures.

<scene name='Papain/Ke_salt_bridges/2'>Salt bridges</scene> strongly contribute to the tertiary structure of papain.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue. The tertiary structure of papain is also maintained by three <scene name='Papain/9pap_sam_disulfides/1'>disulfide bonds</scene>, which connect <scene name='Papain/9pap_sam_disulfides_22-63/1'>Cys-22 to Cys63</scene>, <scene name='Papain/9pap_sam_disulfides_56-95/1'>Cys-56 to Cys-95</scene>, and <scene name='Papain/9pap_sam_disulfides_153-200/1'>Cys-153 to Cys-200</scene><ref name="9PAP PDB" />. These disulfide bonds are likely important in conserving the structural integrity of the enzyme as it operates in extracellular environments at high temperatures.

===Active Site and Substrate Binding===

<scene name='Papain/Substrate_binding_sites/1'>Substrate binding sites.</scene>

===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> 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:

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 Papain's secondary structure is composed of 21% <scene name='Papain/Ke_betasheets/1'>beta sheets</scene> (45 residues comprising 17 sheets) and 25% <scene name='Papain/Ke_alphahelices/1'>alpha helices</scene> (51 residues comprising 7 helices).  The rest of the residues, accounting for over 50% of the enzymes structure, make up ordered non-repetative sequences.<ref name="RSCB PDB">http://www.rcsb.org/pdb/explore/explore.do?structureId=9PAP</ref>  These secondary structures may be traced from the N- to C-terminus by means of <scene name='Papain/Lm_elemental/1'>differential coloration</scene>.  As shown in this scene, the red end begins the protein at the N-terminus, and can be traced through the colors of the rainbow to the blue end at the C-terminus.  These secondary structures form as a result of favorable hydrogen bonding interactions within the polypeptide backbone.  Meanwhile, secondary structures are kept in place by hydrophobic interactions and hydrogen bonds between sidechains of adjacent structures.  For example, the <scene name='Papain/Papain_sam_centralhelix/1'>first helix</scene> (residues 25-42) is maintained as a result of <scene name='Papain/Papain_sb_helix1_hbonds/1'>hydrogen bonds</scene> between backbone carbonyl atoms and the hydrogen on the amide nitrogen four residues away.  However, <scene name='Papain/Papain_sam_nohbondswhelix1/1'>no hydrogen bonds</scene> are present between this helix and the rest of the protein, suggesting that this helix is coordinated primarily by hydrophobic interactions.  This is reasonable given its central location in the enzyme.  As expected, the helix contains many <scene name='Papain/Papain_sb_helix1_hydrophobic/1'>hydrophobic residues</scene> (red residues are hydrophilic).
-<scene name='Papain/Ke_salt_bridges/1'>Salt bridges</scene> strongly contribute to the tertiary structure of papain.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue.  The tertiary structure of papain is also maintained by three <scene name='Papain/9pap_sam_disulfides/1'>disulfide bonds</scene>, which connect <scene name='Papain/9pap_sam_disulfides_22-63/1'>Cys-22 to Cys63</scene>, <scene name='Papain/9pap_sam_disulfides_56-95/1'>Cys-56 to Cys-95</scene>, and <scene name='Papain/9pap_sam_disulfides_153-200/1'>Cys-153 to Cys-200</scene><ref name="9PAP PDB" />.  These disulfide bonds are likely important in conserving the structural integrity of the enzyme as it operates in extracellular environments at high temperatures.
+<scene name='Papain/Ke_salt_bridges/2'>Salt bridges</scene> strongly contribute to the tertiary structure of papain.<ref>http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html</ref> In this particular image, clarification of residue coordination is demonstrated by color: paired residues are shown in the same color, oxygen is shown in red, and nitrogen is shown in blue.  The tertiary structure of papain is also maintained by three <scene name='Papain/9pap_sam_disulfides/1'>disulfide bonds</scene>, which connect <scene name='Papain/9pap_sam_disulfides_22-63/1'>Cys-22 to Cys63</scene>, <scene name='Papain/9pap_sam_disulfides_56-95/1'>Cys-56 to Cys-95</scene>, and <scene name='Papain/9pap_sam_disulfides_153-200/1'>Cys-153 to Cys-200</scene><ref name="9PAP PDB" />.  These disulfide bonds are likely important in conserving the structural integrity of the enzyme as it operates in extracellular environments at high temperatures.
 ===Active Site and Substrate Binding===
+<scene name='Papain/Substrate_binding_sites/1'>Substrate binding sites.</scene>
 ===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>  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: