Sandbox Reserved 1851: Difference between revisions
From Proteopedia
Jump to navigationJump to search
Micah Zile (talk | contribs) No edit summary |
Micah Zile (talk | contribs) No edit summary |
||
| Line 1: | Line 1: | ||
{{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | {{Sandbox_Reserved_CH462_Biochemistry_II_2025}}<!-- PLEASE ADD YOUR CONTENT BELOW HERE --> | ||
==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design== | ==Novel Diels-Alder Catalyst Identified Using ''de novo'' Design== | ||
<Structure load='4o5t' size='350 | <Structure load='4o5t' size='350' align='right' caption='Diels-Alderase shown as cartoon with substrate analog bound' scene='10/1075253/Front_page/2 | ||
' /> | ' /> | ||
==Introduction== | ==Introduction== | ||
| Line 9: | Line 9: | ||
In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO. | In the active state, there are two catalytic <scene name='10/1075253/Active_site_residues/6'>residues</scene> that aim to stabilize the transition state of the Diels-Alder reaction. Y134 acts as a <scene name='10/1075253/Ligand/5'>hydrogen bond donor</scene> to the oxygen on the <scene name='10/1075253/Ligand/2'>ligand</scene>. Q208 acts as a <scene name='10/1075253/Ligand/4'>hydrogen bond acceptor</scene> to the nitrogen on the ligand as well as a donor to the neighboring oxygen. These interactions help reduce the energetic gap between orbitals allowing the reaction to proceed, outlined in HOMO/LUMO. | ||
===Helix Addition=== | ===Helix Addition=== | ||
In the evolution process, researchers added a 16-residue a-helix motif to the top of the binding site. The hydrophobic helix “functions as a lid to constrain the substrates in a productive orientation for reaction” (CITE), decreasing the Km of the enzyme and increasing the catalytic efficiency, as seen in the measured kinetics of the enzyme. | |||
==Mechanism== | ==Mechanism== | ||
===Uncatalyzed Reaction=== | ===Uncatalyzed Reaction=== | ||
===Orbital Stabilization=== | ===Orbital Stabilization=== | ||
==Structural Details== | ==Structural Details== | ||
===Active Site=== | ===Active Site=== | ||
| Line 22: | Line 21: | ||
DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues. | DA_20_10 provided key mutations in and around the active site that increased the hydrophobicity, provided structural stability, and increased interactions between the ligand and surrounding residues. | ||
====Q162R==== | ====Q162R==== | ||
Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction. | Residue 162 resides near the top of the binding entrance to the enzyme, and is within 3A in most models on the enzyme. It can act as a hydrogen bond donor to the terminal phosphate on the ligand when in proximity. To increase this interaction, the group chose a Q to R mutation, which decreased the length of the potential hydrogen bond to within 2.5A, increasing the strength of the interaction. | ||
====S284A==== | ====S284A==== | ||
Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues. | Residue 284 resides deep within the binding pocket of the enzyme. The group chose this mutation to increase the hydrophobicity of the binding pocket and reduce reactivity, without also introducing steric hindrance near the catalytic residues. | ||
====A285N==== | ====A285N==== | ||
Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. | Residue 285, as follows, is also buried within the binding pocket. The group introduced this mutation to increase steric hindrance with the catalytic tyrosine, reducing the number of rotamers the residue has to increase the reactivity of the enzyme by lowering the distance between Y134 and the ligand. | ||
== Relevance == | == Relevance == | ||
===Chemical Applications=== | ===Chemical Applications=== | ||
===Improvements=== | ===Improvements=== | ||
This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. | This is a sample scene created with SAT to <scene name="/12/3456/Sample/1">color</scene> by Group, and another to make <scene name="/12/3456/Sample/2">a transparent representation</scene> of the protein. You can make your own scenes on SAT starting from scratch or loading and editing one of these sample scenes. | ||
