Putting enzymes on pause
of 0.5 µM, 20 µL of reaction buffer, and pNPP at a concentration of 10 mM. Triplicates of both a maximum and minimum activity control were made, with the maximum activity control containing the enzyme and substrate without the inhibitor, representing the highest possible enzyme activity. In contrast, the minimum activity control only includes the substrate (pNPP), representing the lowest possible enzyme activity (close to 0%). The inhibition percentage of each candidate inhibitor was then calculated. Inhibitor design . A docked positioning of the inhibitor with the highest percentage inhibition was modelled in MOE using the Amber14:EHT force field. Then, the inhibitor was edited by both the addition and subtraction of functional groups. The changes followed Lipinski’s rule of 5, meaning that there could be a maximum of 5 hydrogen bond donors and 10 hydrogen bond acceptors, a maximum molecular weight of 500 Da, and a log P (measuring lipophilicity) below 5. Finally, the positioning of the inhibitor was minimized in order to calculate affinity. The inhibitors were designed based on their affinity for the active site and the extent to which they entirely blocked the site.
RESULTS
Figure 1. A digital image of the gel electrophoresis results helps deduce the molecular mass of the enzymes. As time went on, the protein became purer. The protein is in columns 7 and 9 and has a molecular weight between 35.5 and 36, as the most significant band percentages were at 35.5 and 35.7.
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