Table 1 provides the statistics for the first part of lab 11. In this section we used a 1:5 dilution of AdhP from our elution two. Using a spectrophotometer set to 280nm, the concentration of this dilution was found to be 0.08725uM and would be mixed with the various amounts of EtOH and NAD+. When mixed we were then allowed to observe the kinetics using spectroscopy set at 340nm. The data can be seen in Table 1 and Table 2. We were the able to plot this data using Excel thus creating Figures 1 and 2. With these figures and help of solver, we were able to calculate and record our Vmax, Km, and kcat for both EtOH and NAD+ in the presence of the enzyme AdhP as seen in Table 4. When looking at the data set as a whole once can see some possible
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Table 5 provides information regarding the changing amounts of substrate and inhibitor (not concentration), and how this affected V. From the results in Table 5, we were able to create Figure 3. Figure 3 represents a Lineweaver-Burk plot and was used to determine the type of inhibition occurring. In an ideal situation a Lineweaver-Burk plot would look like Figure 6. Even though there seems to be some error occurring, it looks as if pyrazole acted as a competitive inhibitor. This means pyrazole bind to the active site, where normally the substrate would bind, on AdhP. When this happens the reaction is prevented, but can be overcome by adding more substrate. This type of inhibition can be seen in Figure 7. Figure 3 then allowed us to create a Dixon Plot as seen in Figure 4. This Figure showed the largest source of error and actually had a negative slope. A positive slope was expected, thus making our calculation for -Ki rather difficult. –Ki represents the dissociation constant for the complexes created in this experiment (enzyme-substrate and enzyme-substrate-inhibitor) and using the x-intercept of Figure 4 we determined that this value was -1.8081849. This value, as shown in Table 6, should have been a positive number, but due to errors that did not occur. All of the value seen in Table 6 helped create Figure 4.The errors from both graphs and tables were presumably due to the same errors listed above within lab
Table 5 provides information regarding the changing amounts of substrate and inhibitor (not concentration), and how this affected V. From the results in Table 5, we were able to create Figure 3. Figure 3 represents a Lineweaver-Burk plot and was used to determine the type of inhibition occurring. In an ideal situation a Lineweaver-Burk plot would look like Figure 6. Even though there seems to be some error occurring, it looks as if pyrazole acted as a competitive inhibitor. This means pyrazole bind to the active site, where normally the substrate would bind, on AdhP. When this happens the reaction is prevented, but can be overcome by adding more substrate. This type of inhibition can be seen in Figure 7. Figure 3 then allowed us to create a Dixon Plot as seen in Figure 4. This Figure showed the largest source of error and actually had a negative slope. A positive slope was expected, thus making our calculation for -Ki rather difficult. –Ki represents the dissociation constant for the complexes created in this experiment (enzyme-substrate and enzyme-substrate-inhibitor) and using the x-intercept of Figure 4 we determined that this value was -1.8081849. This value, as shown in Table 6, should have been a positive number, but due to errors that did not occur. All of the value seen in Table 6 helped create Figure 4.The errors from both graphs and tables were presumably due to the same errors listed above within lab