Sandbox Reserved 1852: Difference between revisions
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==Introduction== | ==Introduction== | ||
The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using de novo enzyme design, which relies on computational modeling that is refined through programming collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/> | The Diels Alderase aims to catalyze the Diels-Alder reaction for use in synthetic organic chemistry. Specifically, the enzyme surpasses uncatalyzed reactions by generating a product that is entirely [https://en.wikipedia.org/wiki/Stereoselectivity#:~:text=In%20chemistry%2C%20stereoselectivity%20is%20the,of%20a%20pre%2Dexisting%20one. stereoselective] for the 3R,4S endo form. The Diels-Alderase was built using ''de novo'' enzyme design, which relies on computational modeling that is refined through programming collaborative problem-solving from online users. The original protein was made using the [https://en.wikipedia.org/wiki/Rosetta@home Rosetta] computational design program, where a potential active site was built and tested against a library of scaffold proteins. Later, as the active site was perfected, future generations of the Diels-Alderase were made using an online protein folding game called [https://en.wikipedia.org/wiki/Foldit Foldit,] where players competed to improve binding efficiency by completing various challenges.<ref name="Eiben"/> | ||
[[Image:DielsAlderasesubstrates.png| | [[Image:DielsAlderasesubstrates.png|350px|left|thumb|Figure 1. Diels-Alderase substrates. Diene is 4-carboxybenzyl trans-1,3-butadiene-1-carbamate; dienophile is N,N- dimethylacrylamide.]] | ||
The Diels-Alderase | The Diels-Alderase was designed to connect a diene and dienophile in a [https://en.wikipedia.org/wiki/Diels%E2%80%93Alder_reaction Diels-Alder reaction.] It accomplishes this by decreasing the energy gap between the dienophile’s lowest unoccupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (LUMO)] and the diene’s highest occupied molecular orbital [https://en.wikipedia.org/wiki/HOMO_and_LUMO (HOMO)] in the transition state.<ref name="Siegel">PMID:20647463</ref> The current most active form of the Diels-Alderase is modelled under the PDB code [https://www.rcsb.org/structure/4O5T 4o5t]. | ||
The binding pocket of 4O5T is selective for two substrates, 4-carboxybenzyl trans-1,3-butadiene-1-carbamate ([https://en.wikipedia.org/wiki/Diene diene]) and N,N- dimethylacrylamide (dienophile). These substrates are shown as a single, combined ligand–name—in the protein model. The binding site contains a [https://en.wikipedia.org/wiki/Hydrogen_bond hydrogen bond] donor (Y134) which lowers the LUMO energy and stabilizes the negative charge on the dienophile and a hydrogen bond acceptor (Q208) that increases the HOMO energy and stabilizes the positive charge on the diene.<ref name="Siegel"/> Both of these H-bonding interactions work to stabilize the transition state, while also orienting the substrates in optimal conformations for reacting. | |||
Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.] | Overall, the Diels-Alderase stimulates improvement in synthetic laboratories and demonstrates early success in the now-prominent world of [https://www.nobelprize.org/prizes/chemistry/2024/press-release/ computational enzyme design.] | ||
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==Kinetics== | ==Kinetics== | ||
[[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure X. A | [[Image:DAcombinedkineticdata Large.jpeg|700px|left|thumb|Figure X. A) Catalytic efficiencies of key Diels-Alderase generations. Kinetic data was measured at 25 degrees Celsius, in PBS, at pH 7.4. B) Improvement of catalytic efficiency across generations.<ref name="Preiswerk">PMID:24847076</ref>]] | ||
Classic [https://en.wikipedia.org/wiki/Michaelis%E2%80%93Menten_kinetics Michaelis-Menten kinetics]were determined for each generation of the enzyme. As the Diels-Alderase relies on a catalyzed interaction between both the diene and dienophile, a Michaelis binding constant (''Km'' value) was determined for each substrate separately before catalytic efficiency was calculated. The CE20 model of the enzyme is over 300-fold more efficient than the first enzyme model due to increasing active site specificity.<ref name="Preiswerk"/> | |||
