Sandbox 30: Difference between revisions
From Proteopedia
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 6: | Line 6: | ||
==Overview== | ==Overview== | ||
<applet load='9PAP' size=' | <applet load='9PAP' size='400' frame='true' align='right' scene='Sandbox_30/Papain_default/7' caption='Click on the links to the left to view different structural aspects. PDB code for this 1.65 Å resolution structure is 9PAP.' /> | ||
Papain is a 23.4 kDa, 212 residue cysteine endopeptidase originating from the fruit of ''Carica papaya'', where it is present in significant amounts along with three other cysteine proteases, chymopapain, glycyl endopeptidase, and caricain<ref name="9PAP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP] 9PAP PDB</ref><ref name="sigma">[http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html] Sigma Aldrich</ref><ref name="worthington">[http://www.worthington-biochem.com/pap/default.html] Worthington Biochemical Corporation</ref>. Its action was first described by G.C. Roy in 1873. It was studied intensively from the 1950s to the 1960s, during which time it became the second enzyme ever to have its structure determined by x-ray crystallography. Finally, high resolution structural analysis in the 1980s allowed an accurate description of the enzymes active site<ref name="worthington" />. As an enzyme, papain displays very wide hydrolase activity, serving as a general amidase and esterase in addition to its protease activity. As a protease, papain can hydrolyze bonds of basic amino acids, leucine, and glycine. It shows preference for residues preceded by a large hydrophobic residue, but will not cleave is valine is present on the carboxyl side of a potential cleavage site. In addition to being a very non-specific enzyme, papain is also unusually heat resistant, with maximal activity occurring at a temperature of 65° C. These properties have led to use of papain in a large variety areas. One of these areas is biological research, were papain is utilized in cell isolation. It is also useful in immunological techniques because of its ability to cleave the connection between the crystallizable fragment domain and the immunoglobulin domain of antibodies<ref name="sigma" />. Papain has also found use as an inflammation control agent, a digestive aid, and even a meat tenderizer<ref>[http://www.webmd.com/vitamins-supplements/ingredientmono-69-PAPAIN.aspx?activeIngredientId=69&activeIngredientName=PAPAIN] WebMD</ref>. | Papain is a 23.4 kDa, 212 residue cysteine endopeptidase originating from the fruit of ''Carica papaya'', where it is present in significant amounts along with three other cysteine proteases, chymopapain, glycyl endopeptidase, and caricain<ref name="9PAP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=9PAP] 9PAP PDB</ref><ref name="sigma">[http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/analytical-enzymes/papain.html] Sigma Aldrich</ref><ref name="worthington">[http://www.worthington-biochem.com/pap/default.html] Worthington Biochemical Corporation</ref>. Its action was first described by G.C. Roy in 1873. It was studied intensively from the 1950s to the 1960s, during which time it became the second enzyme ever to have its structure determined by x-ray crystallography. Finally, high resolution structural analysis in the 1980s allowed an accurate description of the enzymes active site<ref name="worthington" />. As an enzyme, papain displays very wide hydrolase activity, serving as a general amidase and esterase in addition to its protease activity. As a protease, papain can hydrolyze bonds of basic amino acids, leucine, and glycine. It shows preference for residues preceded by a large hydrophobic residue, but will not cleave is valine is present on the carboxyl side of a potential cleavage site. In addition to being a very non-specific enzyme, papain is also unusually heat resistant, with maximal activity occurring at a temperature of 65° C. These properties have led to use of papain in a large variety areas. One of these areas is biological research, were papain is utilized in cell isolation. It is also useful in immunological techniques because of its ability to cleave the connection between the crystallizable fragment domain and the immunoglobulin domain of antibodies<ref name="sigma" />. Papain has also found use as an inflammation control agent, a digestive aid, and even a meat tenderizer<ref>[http://www.webmd.com/vitamins-supplements/ingredientmono-69-PAPAIN.aspx?activeIngredientId=69&activeIngredientName=PAPAIN] WebMD</ref>. | ||
| Line 24: | Line 24: | ||
==Catalytic Mechanism== | ==Catalytic Mechanism== | ||
<applet load='1pop' size=' | <applet load='1pop' size='300' frame='true' align='right' scene='Sandbox_30/Papain_ligand_default/3' caption='Papain crystallized with substrate analog leupeptin (green) covalently bound to the catalytic Cys-25. The PBD code for this structure is 1POP' /> | ||
Like serine proteases, cysteine proteases contain a <scene name='Sandbox_30/Papain_ligand_active-site/2'>catalytic triad</scene> of residues. In the case of papain, these residues are Cys-25, His-159, and Arg-175. Papain also contains a fourth residue, <scene name='Sandbox_30/Papain_ligand_active-sitegln19/4'>Gln-19</scene> that has been shown to play an important role in catalysis and is likely involved in the formation of the oxyanion hole. 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 the oxyanion hole. 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 Cys-25 sulfur acts as a leaving group, releasing the N-terminal portion of the peptide and regenerating the enzyme<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>. | Like serine proteases, cysteine proteases contain a <scene name='Sandbox_30/Papain_ligand_active-site/2'>catalytic triad</scene> of residues. In the case of papain, these residues are Cys-25, His-159, and Arg-175. Papain also contains a fourth residue, <scene name='Sandbox_30/Papain_ligand_active-sitegln19/4'>Gln-19</scene> that has been shown to play an important role in catalysis and is likely involved in the formation of the oxyanion hole. 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 the oxyanion hole. 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<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| | [[Image:Papainmech6.jpg|200px|left|thumb| General mechanism of papain catalysis<ref>[http://chemistry.umeche.maine.edu/CHY431/Peptidase10.html] University of Maine</ref>. Arg-175, which orients His 159, and Gln-19, which contributes to the formation of the oxyanion hole, are not shown.]] | ||
===Substrate Binding=== | |||
Papain interacts with the covalently bound leupeptin in a variety of ways. Several residues of the enzyme and a few methanol molecules form <scene name='Sandbox_30/Papain_ligand_h-bond_ints/1'>hydrogen bonds</scene> with the ligand (starred). However, the vast majority of the enzyme-ligand interactions are <scene name='Sandbox_30/Papain_ligand_hydrophobics/1'>hydrophobic</scene>. Leupeptin is coordinated by a variety of tyrosine, tryptophan, and valine residues<ref name="1POP PDB">[http://www.pdb.org/pdb/explore/explore.do?structureId=1pop] 1POP PDB</ref>. | |||
==References== | |||
<references /> | |||
