Sandbox 30: Difference between revisions
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==Catalytic Mechanism== | ==Catalytic Mechanism== | ||
<applet load='1pop' size='300' frame='true' align='right' scene='Sandbox_30/Papain_ligand_default/ | <applet load='1pop' size='300' frame='true' align='right' scene='Sandbox_30/Papain_ligand_default/4' caption='Papain crystallized with substrate analog leupeptin (blue) covalently bound to the catalytic Cys-25. The PBD code for this structure is 1POP' /> | ||
[[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.]] | [[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.]] | ||
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>. | 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>. | ||
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===Substrate Binding=== | ===Substrate Binding=== | ||
In order to investigate binding of protein substrates to papain, the enzyme was crystallized with the broad-spectrum competitive protease inhibitor leupeptin, shown in blue in the ribbon diagram. It has the structure Ac-Leu-Leu-Arginal, where Ac is an acetyl group attached to the nitrogen of the first leucine. The inhibitor functions by binding to the enzyme's active site, where the catalytic nucleophile (cysteine in this case) attacks the arginal aldehyde. This forms a tight-binding transition state from which the normal catalytic mechanism cannot proceed, due to this carbonyl having no potential leaving groups attached to it. Analysis of the resulting structure revealed that the | |||
<scene name='Sandbox_30/Papain_inhibitor_activesite/3'>substrate binding pocket</scene> of papain consists primarily of a variety of hydrophobic residues, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make hydrogen bonds with some of the leupeptin atoms, most notably the arginal oxyanion. | |||
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>. | |||
19,25,158,66,67,68,133,57,61,26,160,157 | |||
hydrophobics start at 66 | |||
==References== | ==References== | ||
<references /> | <references /> | ||
