Sandbox 30: Difference between revisions

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==Catalytic Mechanism==

[[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/3'>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-sitegln19/5'>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===

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 papain) 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 bonded 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 <scene name='Sandbox_30/Papain_inhibitor_hydrophobics/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make <scene name='Sandbox_30/Papain_inhibitor_h-bonds/2'>hydrogen bonds</scene> with some of the leupeptin atoms. These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both ~~Gln19~~ and the amide nitrogen of the catalytic ~~Cys25~~ with the arginal carbanion, forming the catalytically important oxyanion hole. In addition, ~~Gly66~~ interacts with the second leucine in leupeptin while ~~Asp158~~ interacts with a hydrogen on the arginal. These interaction further stabilize and orient the substrate in the binding pocket.

<scene name='Sandbox_30/Papain_inhibitor_activesite/3'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='Sandbox_30/Papain_inhibitor_hydrophobics/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make <scene name='Sandbox_30/Papain_inhibitor_h-bonds/2'>hydrogen bonds</scene> with some of the leupeptin atoms. These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both Gln-19 and the amide nitrogen of the catalytic Cys-25 with the arginal carbanion, forming the catalytically important oxyanion hole. In addition, Gly-66 interacts with the second leucine in leupeptin while Asp-158 interacts with a hydrogen on the arginal. These interaction further stabilize and orient the substrate in the binding pocket<ref name="Schroder">[http://www.sciencedirect.com/science/article/pii/001457939381128M] Schröder, E., C. Phillips, E. Garman, K. Harlos, C. Crawford. 1997. X-ray crystallographic structure of a papain-leupeptin complex. FEBS Letters 315: 38-42</ref>.

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@@ Line 25: / Line 25: @@
 ==Catalytic Mechanism==
-<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'  />
+<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.]]
-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/3'>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-sitegln19/5'>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>.
@@ Line 34: / Line 34: @@
 ===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 papain) 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 bonded 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 <scene name='Sandbox_30/Papain_inhibitor_hydrophobics/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin.  Some of the enzyme's residues also make <scene name='Sandbox_30/Papain_inhibitor_h-bonds/2'>hydrogen bonds</scene> with some of the leupeptin atoms.  These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both Gln19 and the amide nitrogen of the catalytic Cys25 with the arginal carbanion, forming the catalytically important oxyanion hole.  In addition, Gly66 interacts with the second leucine in leupeptin while Asp158 interacts with a hydrogen on the arginal.  These interaction further stabilize and orient the substrate in the binding pocket.
+<scene name='Sandbox_30/Papain_inhibitor_activesite/3'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='Sandbox_30/Papain_inhibitor_hydrophobics/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin.  Some of the enzyme's residues also make <scene name='Sandbox_30/Papain_inhibitor_h-bonds/2'>hydrogen bonds</scene> with some of the leupeptin atoms.  These hydrogen bonds, shown in yellow, include interactions between both hydrogens on both Gln-19 and the amide nitrogen of the catalytic Cys-25 with the arginal carbanion, forming the catalytically important oxyanion hole.  In addition, Gly-66 interacts with the second leucine in leupeptin while Asp-158 interacts with a hydrogen on the arginal.  These interaction further stabilize and orient the substrate in the binding pocket<ref name="Schroder">[http://www.sciencedirect.com/science/article/pii/001457939381128M] Schröder, E., C. Phillips, E. Garman, K. Harlos, C. Crawford. 1997. X-ray crystallographic structure of a papain-leupeptin complex. FEBS Letters 315: 38-42</ref>.