Papain: Difference between revisions
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Papain is a relatively simple enzyme, consisting of a single 212 residue chain. A majority of papain's residues are <scene name='Papain/Hydrophobicity_papain/1'>hydrophobic</scene> as shown in purple. As with all proteins, it is primarily the exclusion of these residues by water that leads to papain's assumption of a globular form. Despite its apparent simplicity and small size, papain folds into two distinct, evenly sized <scene name='Papain/Sk_two_domains/2'>domains</scene>, each with its own <scene name='Papain/Sk_hydrophobic_core/2'>hydrophobic core</scene> (surface residues are transparent, hydrophobic-core residues are colored and opaque, and the remaining are polar, non-surface residues).<ref name="Structure">PMID:6502713</ref> | Papain is a relatively simple enzyme, consisting of a single 212 residue chain. A majority of papain's residues are <scene name='Papain/Hydrophobicity_papain/1'>hydrophobic</scene> as shown in purple. As with all proteins, it is primarily the exclusion of these residues by water that leads to papain's assumption of a globular form. Despite its apparent simplicity and small size, papain folds into two distinct, evenly sized <scene name='Papain/Sk_two_domains/2'>domains</scene>, each with its own <scene name='Papain/Sk_hydrophobic_core/2'>hydrophobic core</scene> (surface residues are transparent, hydrophobic-core residues are colored and opaque, and the remaining are polar, non-surface residues).<ref name="Structure">PMID:6502713</ref> | ||
These two subunits are held together with <scene name='Papain/Armcrossing/1'>"arm" linkage</scene>, where each protein domain holds the opposite domain. In papain's case the "arm" crossing primarily occurs on or near the surface.<ref>[http://kinemage.biochem.duke.edu/teaching/anatax/html/anatax.2i.html] Jane S. Richardson</ref> It is between these two domains that the <scene name='Papain/9pap_bindingpocket_wrtdomains/4'>substrate binding pocket</scene> is situated. The two domains interact with one another via hydrophobic interactions, <scene name='Papain/Twodomainshbonds/2'>hydrogen bonds</scene> (shown in white), and electrostatic interactions in this cleft. For example, <scene name='Papain/Sk_domain_contact_valine/ | These two subunits are held together with <scene name='Papain/Armcrossing/1'>"arm" linkage</scene>, where each protein domain holds the opposite domain. In papain's case the "arm" crossing primarily occurs on or near the surface.<ref>[http://kinemage.biochem.duke.edu/teaching/anatax/html/anatax.2i.html] Jane S. Richardson</ref> It is between these two domains that the <scene name='Papain/9pap_bindingpocket_wrtdomains/4'>substrate binding pocket</scene> is situated. The two domains interact with one another via hydrophobic interactions, <scene name='Papain/Twodomainshbonds/2'>hydrogen bonds</scene> (shown in white), and electrostatic interactions in this cleft. For example, <scene name='Papain/Sk_domain_contact_valine/3'>Valine-32</scene> from the L Domain hydrophobically interacts with the carbon atoms on residues Lys-174, Ala-162 and Pro-129 of the R Domain. <scene name='Papain/Sk_domain_contact_glutamine/4'>Gln-19</scene> hydrogen bonds multiple times with the oxygen atoms of Ser-176 and also with the oxygen atom on Tyr88. Electrostatic interactions are seen between <scene name='Papain/Sk_domain_contact_glu_and_lys/4'>Glu-35 and Lys-174</scene> where the carboxyl group of Glu-35 forms an ionic bond with the ammonia group of the Lys-174 residue. The <scene name='Papain/Twodomainsallncbonds/1'>sum total</scene> of interactions within the cleft between the two domains ensures that the lobes do not move with respect to one another. <ref>[http://books.google.com/books?hl=en&lr=&id=fk1hbZdPTEgC&oi=fnd&pg=PA79&dq=aromatic+residues+in+papain&ots=L8SvlkQaZU&sig=xZ2l8kj52PD7DzuiAQ1zah0CU2M#v=onepage&q=aromatic%20residues%20in%20papain&f=false] The Structure of Papain </ref> | ||
In addition to hydrophobic residues, papain contains a variety of <scene name='Papain/Sk_polar_residues/2'>polar residues</scene>, some carrying a <scene name='Papain/Sk_acidic_residues/2'>negative charge</scene>, shown in gray at physiological pH, and are therefore acidic; others a <scene name='Papain/Sk_basic_residues/2'>positive charge</scene>, shown in purple, and are therefore basic. The rest of the <scene name='Papain/Sk_basic_residues/3'>polar residues</scene>, shown in a light gray, are neutral. As expected, the charged <scene name='Papain/Termini/2'>termini</scene> face outward due to their hydrophilic nature. | In addition to hydrophobic residues, papain contains a variety of <scene name='Papain/Sk_polar_residues/2'>polar residues</scene>, some carrying a <scene name='Papain/Sk_acidic_residues/2'>negative charge</scene>, shown in gray at physiological pH, and are therefore acidic; others a <scene name='Papain/Sk_basic_residues/2'>positive charge</scene>, shown in purple, and are therefore basic. The rest of the <scene name='Papain/Sk_basic_residues/3'>polar residues</scene>, shown in a light gray, are neutral. As expected, the charged <scene name='Papain/Termini/2'>termini</scene> face outward due to their hydrophilic nature. | ||
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===Substrate Binding=== | ===Substrate Binding=== | ||
Papain has a relatively small <scene name='Papain/Active_site/1'>active site</scene> consisting of three residues: Cys-25, His-159, and | Papain has a relatively small <scene name='Papain/Active_site/1'>active site</scene> consisting of three residues: Cys-25, His-159, and Asn-175. The sulfhydryl group on Cys-25 often forms covalent bonds with substrates. His-159 supports Cys-25, and while Arg-175 does not directly participate in the catalytic mechanism, it keeps histidine-159 in its stabilized imidazole form. In addition to the active site, sometimes referred to as the catalytic site, Papain consists of many <scene name='Papain/Substrate_binding_sites/2'>substrate binding subsites.</scene> Subsites are defined as the regions on the enzyme surface which interact with one amino acid residue of the substrate. In the case of Papain, there are seven subsites labeled as follows: S1, S2, S3, S4, located on the amino side of the catalytic site; S1', S2', and S3', located on the carboxyl side of the catalytic site. The binding sites of a substrate are labeled according to how they fit into the binding cleft. P1 associates with S1, P2 with S2, etc. All seven of Papain's subsites hydrogen bond to the corresponding substrate P subsites. The following <scene name='Papain/Substrate_binding_sites/3'>subsites</scene> of Papain have been identified as follows: S1 - His-159; S2 - Trp-177; S3 - Gln-19; S4 - Gly-23; S2' - Asp-158; and S3' - Asp-64. Unfortunately, S1' has yet to be identified. | ||
The seven subsites of Papain have various preferences for substrate residues. Through a variety of experiments, Berger & Schechter<ref>PMID:4399049</ref>, were able to show that S1 binds alanine better than glycine, and the larger side chains of lysine, arginine, leucine, and phenylalanine better than alanine. Thus showing that binding in S1 is predominantly hydrophobic. S2 prefers a phenylalanine or a valine residue. Interestingly enough, S2 binds to hydrophobic residues of both short and long peptide chains. They were able to show that subsites S1' and S2' are strongly stereospecific. The conclusion of their research was that Papain's binding site residues show a strong stereospecificity, special interactions, and space limitations.<ref>PMID:4399049</ref> | The seven subsites of Papain have various preferences for substrate residues. Through a variety of experiments, Berger & Schechter<ref>PMID:4399049</ref>, were able to show that S1 binds alanine better than glycine, and the larger side chains of lysine, arginine, leucine, and phenylalanine better than alanine. Thus showing that binding in S1 is predominantly hydrophobic. S2 prefers a phenylalanine or a valine residue. Interestingly enough, S2 binds to hydrophobic residues of both short and long peptide chains. They were able to show that subsites S1' and S2' are strongly stereospecific. The conclusion of their research was that Papain's binding site residues show a strong stereospecificity, special interactions, and space limitations.<ref>PMID:4399049</ref> | ||
