Lab 10: DATA ANALYSIS

Your data will be provided by your TAs after you complete the in-lab activities.

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 – Velocity as a Function of Substrate” choose “Michaelis-Menten.”
    • Go into the “Compare” tab to have Prism compare the K_M for wild type versus mutant.
    • Go to the “Confidence” tab and select “Symmetrical (asymptotic) approximate CI” then select “Show SE of parameters.” Press OK.
• You should now have values for V_max and K_M with standard errors, a determination of statistical significance for the K_M including a p-value, and a plot showing the fit.
  • Also make note of the “Degrees of freedom” provided for both wt and mutant. You will need this number in a future analysis.
• 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 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)

• You are manipulating values that all have their own associated error. You will need to report standard error values for k_cat 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
• Once you have determined the standard error for k_cat for both wild type and mutant, you can utilize a different type of Prism analysis to determine p-values.
• Open Prism and select from the far left menu “Column” (by contrast, the default is “XY”)
• Select “Enter and plot error values already calculated elsewhere” and from the dropdown menu choose “Mean, SEM, N”

• Enter the following for wt and mutant:
  • Mean: the k_cat values you determined
  • SEM: the standard error of the k_cat values (determined by propagation of error)
  • N: the degrees of freedom (determined by Prism above) + 1
• Go to “Analyze” and select, under Column analyses, “t tests (and nonparametric tests.” Press OK. (Leave all the parameters and options as the default.)
• The Results sheet should provide a p-value.

k_cat /K_M Calculation:

• Now take the k_cat values you calculated and divide them by K_M to determine the catalytic efficiency.
• You will again need to use principles of propagation of error to determine standard errors for each k_cat /K_M value.
• Repeat the Prism analysis above to determine a p-value for k_cat /K_M.