Lab 10: DATA ANALYSIS

Complete the following steps to calculate V_max, K_M, k_cat and k_cat/K_M for wt and mutant HCAII.

• Make a table of of initial velocities (in µM product/time) and substrate concentrations. To calculate µM product/time, you need to know: The molar absorption coefficient of PNP is 1.73 x 10⁴ M^-1cm^-1 at 405 nm, and the effective path length (b) of the spectrophotometer cuvettes is 1.0 cm.

Michaelis-Menten Fit:

• Open GraphPad Prism and select “Enter 3 replicate values in side-by-side subcolumns,” hit “Create”
• Paste your data in. You can put replicates in adjacent columns.
• Select “Analyze”
  • Under “XY analyses” select “Nonlinear regression (curve fit)” Press OK
  • Under “Enzyme kinetics – Substrate vs Velocity” choose “Michaelis-Menten.”
    • Go into the “Compare” tab to have Prism compare the V_max for wild type versus mutant.
      • Note that if you used different enzyme concentrations for wt and mutant, comparing V_max is not valid. In this case, you will still use Prism to calculate V_max values, but you cannot use the determination of significance from Prism. Instead, you need to convert V_max into k_cat, which can then be directly compared.
    • Update for Prism 8: Go to the “Confidence” tab and select “Symmetrical (asymptotic) approximate CI” then select “Show SE of parameters.” Press OK.
      • Prism doesn’t recommend this fit, but in order to easily propagate the error associated with V_max later, we need a Standard Error. Based on a few initial trials, this type of CI calculation only differs very slightly from the recommended type.
• Go back to the data table and again select “Analyze” and “Nonlinear regression (curve fit)”.
  • A window should pop up asking where to put this new analysis, select “Analyze this table again, creating a new results sheet.”
  • Repeat the fit as described above, but this time select to compare K_M for wild type versus mutant (instead of V_max). Again go into the Confidence tab to show standard error. Press OK
• You should now have values for V_max and K_M with standard errors, a determination of statistical significance including a p-value, and a plot showing the fit.
• Note that you also now have a plot of V_o vs [S]. You can play with the plot in Prism to make it look nice, just as you would in Excel: label axes, remove title, add error bars, etc.

 K_cat Calculation:

• Calculate k_cat and k_cat/K_M for your wt and mutant enzymes based on your Michaelis-Menten fit using Equation 3. Pay attention to units: the units of k_cat are sec^-1.

(3)

• For the k_cat calculations, you are manipulating values that all have their own associated error. You will need to report error on k_cat and k_cat/K_M using the principles of propagation of error. Refer to the Appendix for additional help (link opens in a new tab): Appendix 2: Statistical methods used in 551 data analyses