Sandbox Reserved 1852: Difference between revisions
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During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> | During initial computer modelling, over one million potential Diels-Alderase active sites were matched to potential protein scaffolds.<ref name="Siegel"/> Only 2 proteins proved to be sufficiently active after [https://en.wikipedia.org/wiki/Liquid_chromatography%E2%80%93mass_spectrometry LC-MS] screening. DA_20_00, which used a beta-propeller scaffold, had the most success in further mutations and therefore became the Diels-Alderase of choice.<ref name="Siegel"/> However, this initial enzyme's active site had very little catalytic activity, seen in its low catalytic efficiency after kinetic screening [Fig. 4].<ref name="Siegel"/><ref name="Preiswerk"/> | ||
===DA_20_10=== | ===DA_20_10=== | ||
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 [https://en.wikipedia.org/wiki/hydrophobe?variant=zh-tw hydrophobicity], provided structural stability, and increased interactions between the ligand and surrounding residues. | ||
=====Q162R===== | =====Q162R===== | ||
:Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand 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, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/> | :Glu162, a <scene name='10/1075254/Q162/5'>glutamine</scene>, resides near the top of the binding site, and is about than 3Å from the ligand 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, Glu162 was mutated to an <scene name='10/1075254/Q_to_r/2'>arginine</scene>, which decreased the length of the potential hydrogen bond to within 2.5Å in most models, increasing the strength of the interaction.<ref name="Siegel"/> | ||
