Papain: Difference between revisions
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==='''Leupeptin'''=== | ==='''Leupeptin'''=== | ||
Leupeptin is a commonly studied inhibitor of proteases. It inhibits by binding and interacting with the active site which allows it to block the enzyme's desired protein substrate. There are many <scene name='Sandbox_31/1popligand_contacts/1'>residues</scene> that interact with Leupeptin in the active site. The predominant interaction is from hydrophobic interactions between Leupeptin and <scene name='Sandbox_31/1pophydrointeract/1'>active site residues</scene>. In addition to hydrophobic interactions, there are also some hydrogen bonding interactions to hold Leupeptin in the active site of papain. Leupeptin works well at blocking papain from its enzymatic duties. A recent study has shown that Leupeptin actually forms a covalent bond between its <scene name='Sandbox_31/Leupeptin_active/1'>Carbonyl Carbon</scene> and CYS 25. In addition, the residues Gln 19 and CYS 25 form <scene name='Sandbox_31/Leupeptin_active/2'>hydrogen bonds</scene> with the Leupeptin molecule. | |||
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>. | 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>. | ||
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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='Papain/Sk_inhibitor_1/1'>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='Papain/Sk_inhibitor_2/1'>active site cleft</scene> are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159). | 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='Papain/Sk_inhibitor_1/1'>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='Papain/Sk_inhibitor_2/1'>active site cleft</scene> are involved in the complex with the inhibtor (Trp177, Ser21, Cys63, Cys25, Asp158 and His159). | ||
There are a small number of <scene name='Papain/Sk_inhibitor_3/1'>direct hydrogen bonds</scene> between stefin B and papain, however there are many more polar interactions mediated by <scene name='Papain/Sk_inhibitor_4/3'>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='Papain/Sk_inhibitor_5/ | There are a small number of <scene name='Papain/Sk_inhibitor_3/1'>direct hydrogen bonds</scene> between stefin B and papain, however there are many more polar interactions mediated by <scene name='Papain/Sk_inhibitor_4/3'>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='Papain/Sk_inhibitor_5/3'>Van der Waals interactions</scene>. <ref> PMID:2347312 </ref> | ||
</StructureSection> | </StructureSection> | ||
