Putting enzymes on pause
Figure 6. Standard curve for sodium phosphate at varying concentrations. A linear regression was performed for the concentrations 400 µM and below, yielding the equation and the coefficient of
determination. Reactions were carried out with 5 µL of sodium phosphate at a selected concentration, 100 µL of Biomol Green reagent, and 45 µL of enzyme reaction buffer. Absorbance values were measured after incubating for 20 minutes. Sodium phosphate concentrations at 0, 25, 50, 100, 200, 400, 600, and 800 µM were used to obtain absorbance values, which were plotted on a graph. The concentrations from 0 to 400 µM exhibited a linear pattern, whereas higher sodium phosphate concentrations deviated from a linear trend. Thus, the standard curve included the data from concentrations at and below 400 µM to generate a linear regression equation and a high coefficient of determination (figure 5). As pNPP is not a natural Cdc14 substrate, a new assay was designed using phosphopeptides. A standard curve was required for this new assay to establish linearity between known concentrations of sodium phosphate and their corresponding absorbance values, which were then used in conjunction with the absorbance values of the phosphopeptides to determine the unknown concentration of products formed.
Figure 7. Overall X-ray crystal structure of the superposed homology model and 5XW4 (yeast enzyme) template sequences, with backbones colour-coded by RMSD. Regions in green represent areas with lower RMSD values, signifying few structural deviations between the compared structures. Regions in white represent areas with higher RMSD values, indicating significant structural deviations and poor similarity.
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