Papain: Difference between revisions

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In order to investigate binding of protein substrates to papain, Schröder et. al. crystallized the enzyme 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='9pap/Papain_sam_1popactivesite/1'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='9pap/1pop_sam_leupeptin_hydrophobic/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin. Some of the enzyme's residues also make <scene name='9pap/1pop_sam_leupeptin_hbonds/1'>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>.

===Cathepsin K===

The goal of research for the development of an inhibitor for <scene name='Sandbox_34/Cathepsink/1'>cathepsin K</scene> is the hope to develop a treatment for osteoporosis. In two different cathepsin K inhibitors, referenced PDB codes <scene name='Sandbox_34/Cathkaldinhibit/1'>1BP4</scene> and <scene name='Sandbox_34/Cathkketoinhibition/1'>1BQI</scene>, it is evident that the inhibitor binds with much closer proximity than that of Clik148. 1BP4 is a cathepsin K inhibitor, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, that inhibits by interacting with 11 different residues on papain: Gln19, Gly20, Ser21, Gly23, Asn64, Gly65, Gln142, Asp158, His159, Trp177, and Trp181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to <scene name='Sandbox_34/Cathkaldinhibitpistacking/2'>ring stacking</scene> between the aromatic ring of the carbobenzyl group on 1BP4, and TRP177 of papain. The inhibition of papain by IBQI, carbobenzyloxy-(L)-leucinyl-(L)leucinyl methoxymethylketone, is quite similar to that of IBP4, although it does not bind quite as tightly. It binds to seven residues of papain: Gln19, Gly23, Gly65, Gln142, His159, Trp177, Trp181. Additionally, it has similar

<scene name='Sandbox_34/Cathkketoinhibitionringstackin/2'>ring-stacking</scene> between the Cbz ring on the inhibitor and Trp 177, though it is more difficult to visualize with the given PDB file.<ref> PMID:9804696 </ref>

==='''Cathepsin L (PDB ID #: 1CVZ)'''===

Cathepsin L is another inhibitor of the papain enzyme. Cathepsin L interacts with the <scene name='Sandbox_31/Cathepl/1'>papain residues</scene> Gln19, Cys25, Gly66, Asp158, and Trp177 by hydrogen bonding them (Cathepsin L is lime green in the model, and the papain residues active in hydrogen bonding with Cathepsin L are highlighted with yellow halos). In addition to hydrogen bonding, hydrophobic interactions exist to exclude water, allowing the papain enzyme and Cathepsin L to associate even closer. Finally, <scene name='Sandbox_31/Stacking/1'>Ring Stacking</scene> between Trp177 and the Capthespin L molecule hold them tightly together.<ref>http://pubs.acs.org/doi/abs/10.1021/ci800085c</ref> Cathepsin L plays a roll in many different diseases including malaria, leishmaniasis, Chagas' disease, African trypanosomiasis, toxoplasmosis, and amoebiasis.<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923042/?tool=pubmed</ref>Some studies show that there is a relation between cathepsins and certain cancers, alzheimer's, and arthritis.<ref>http://www.biomedcentral.com/1472-6807/10/30</ref>

==='''Stefin B'''===

<scene name='Sandbox_36/Papain_inhibitor_space_fill/1'>Stefin B</scene> acts as a competitive inhibitor to cysteine proteases-- it binds tightly but reversibly to the papain active site. Stefin inhibitors are characterized by M<sub>r</sub> of about 11,000, no disulfide bonds and no associated carbohydrates.

In Stefin B, the Gly9 residue along with two hairpin loops form a "wedge" complementary to the active site groove of papain. This wedge makes extensive and tight interactions with papain and a total of 128 intermolecular atom-atom interactions occur. <scene name='Sandbox_36/Papain_inhibitor_wedge_involve/1'>Residue segments</scene> Met6-Pro11, Gln53-Asn59, Gln101-His104 and Tyr124-Phe125 on the wedge all have some interaction to the enzyme though not always direct. All residues from the base and both sides of the <scene name='Sandbox_36/Papain_inhibitor_activ_involve/1'>active site cleft</scene> are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159).

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There are a small number of <scene name='Sandbox_36/Papain_inhibitor_direct_bond/2'>direct hydrogen bonds</scene> between stefin B and papain, however there are many more polar interactions mediated by <scene name='Sandbox_36/Papain_inhibitor_bridges_inter/1'>solvent bridges</scene>. Thirteen solvent molecules bridge polar residues of the enzyme and inhibitor. Seventeen hydrogen bonds are made with a solvent molecule and stefin. Fourteen of these bridges form a papain contact. The rest of the interactions are largely hydrophobic-- involving apolar <scene name='Sandbox_36/Papain_inhibitor_hydro_inter/2'>Van der Waals interactions</scene>. <ref> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC551902/pdf/emboj00233-0254.pdf </ref>

~~===Clik-158===~~

</StructureSection>

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@@ Line 41: / Line 41: @@
 In order to investigate binding of protein substrates to papain, Schröder et. al. crystallized the enzyme 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='9pap/Papain_sam_1popactivesite/1'>substrate binding pocket</scene> of papain consists primarily of a variety of <scene name='9pap/1pop_sam_leupeptin_hydrophobic/1'>hydrophobic residues</scene>, including tyrosine, tryptophan, and valine, which coordinate the bound leuptin.  Some of the enzyme's residues also make <scene name='9pap/1pop_sam_leupeptin_hbonds/1'>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>.
+===Cathepsin K===
+The goal of research for the development of an inhibitor for <scene name='Sandbox_34/Cathepsink/1'>cathepsin K</scene> is the hope to develop a treatment for osteoporosis. In two different cathepsin K inhibitors, referenced PDB codes <scene name='Sandbox_34/Cathkaldinhibit/1'>1BP4</scene> and <scene name='Sandbox_34/Cathkketoinhibition/1'>1BQI</scene>, it is evident that the inhibitor binds with much closer proximity than that of Clik148. 1BP4 is a cathepsin K inhibitor, N-[(benzyloxy)carbonyl]-L-leucyl-N-[(2S)-1-hydroxy-4-methylpentan-2-yl]-L-leucinamide, that inhibits by interacting with 11 different residues on papain: Gln19, Gly20, Ser21, Gly23, Asn64, Gly65, Gln142, Asp158, His159, Trp177, and Trp181. These interactions range from hydrophobic, electrostatic, and hydrogen bonding, to <scene name='Sandbox_34/Cathkaldinhibitpistacking/2'>ring stacking</scene> between the aromatic ring of the carbobenzyl group on 1BP4, and TRP177 of papain. The inhibition of papain by IBQI, carbobenzyloxy-(L)-leucinyl-(L)leucinyl methoxymethylketone, is quite similar to that of IBP4, although it does not bind quite as tightly. It binds to seven residues of papain: Gln19, Gly23, Gly65, Gln142, His159, Trp177, Trp181. Additionally, it has similar
+<scene name='Sandbox_34/Cathkketoinhibitionringstackin/2'>ring-stacking</scene> between the Cbz ring on the inhibitor and Trp 177, though it is more difficult to visualize with the given PDB file.<ref> PMID:9804696 </ref>
 ==='''Cathepsin L (PDB ID #: 1CVZ)'''===
 Cathepsin L is another inhibitor of the papain enzyme.  Cathepsin L interacts with the <scene name='Sandbox_31/Cathepl/1'>papain residues</scene> Gln19, Cys25, Gly66, Asp158, and Trp177 by hydrogen bonding them (Cathepsin L is lime green in the model, and the papain residues active in hydrogen bonding with Cathepsin L are highlighted with yellow halos). In addition to hydrogen bonding, hydrophobic interactions exist to exclude water, allowing the papain enzyme and Cathepsin L to associate even closer.  Finally, <scene name='Sandbox_31/Stacking/1'>Ring Stacking</scene> between Trp177 and the Capthespin L molecule hold them tightly together.<ref>http://pubs.acs.org/doi/abs/10.1021/ci800085c</ref> Cathepsin L plays a roll in many different diseases including malaria, leishmaniasis, Chagas' disease, African trypanosomiasis, toxoplasmosis, and amoebiasis.<ref>http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923042/?tool=pubmed</ref>Some studies show that there is a relation between cathepsins and certain cancers, alzheimer's, and arthritis.<ref>http://www.biomedcentral.com/1472-6807/10/30</ref>
 ==='''Stefin B'''===
 <scene name='Sandbox_36/Papain_inhibitor_space_fill/1'>Stefin B</scene> acts as a competitive inhibitor to cysteine proteases-- it binds tightly but reversibly to the papain active site.  Stefin inhibitors are characterized by M<sub>r</sub> of about 11,000, no disulfide bonds and no associated carbohydrates.
 In Stefin B, the Gly9 residue along with two hairpin loops form a "wedge" complementary to the active site groove of papain.  This wedge makes extensive and tight interactions with papain and a total of 128 intermolecular atom-atom interactions occur.  <scene name='Sandbox_36/Papain_inhibitor_wedge_involve/1'>Residue segments</scene> Met6-Pro11, Gln53-Asn59, Gln101-His104 and Tyr124-Phe125 on the wedge all have some interaction to the enzyme though not always direct.  All residues from the base and both sides of the <scene name='Sandbox_36/Papain_inhibitor_activ_involve/1'>active site cleft</scene> are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159).
@@ Line 56: / Line 57: @@
 There are a small number of <scene name='Sandbox_36/Papain_inhibitor_direct_bond/2'>direct hydrogen bonds</scene> between stefin B and papain, however there are many more polar interactions mediated by <scene name='Sandbox_36/Papain_inhibitor_bridges_inter/1'>solvent bridges</scene>.  Thirteen solvent molecules bridge polar residues of the enzyme and inhibitor.  Seventeen hydrogen bonds are made with a solvent molecule and stefin.  Fourteen of these bridges form a papain contact.  The rest of the interactions are largely hydrophobic-- involving apolar <scene name='Sandbox_36/Papain_inhibitor_hydro_inter/2'>Van der Waals interactions</scene>. <ref> http://www.ncbi.nlm.nih.gov/pmc/articles/PMC551902/pdf/emboj00233-0254.pdf </ref>
-===Clik-158===
 </StructureSection>