Exercise: Enzyme kinetics in Prism

Go to parent GraphPad Prism statistical analyses

The kinetics of lysozyme was measured as a function of concentration of its substrate NAM-NAG, a disaccharide found as a major structural component in many bacterial cell walls. Lysozyme will hydrolyze the bond between the two sugar components of this disaccharide.
The assay is done as follows: the enzyme produces product at an initial rate that is approximately linear for a short period after the start of the reaction. As the reaction proceeds and substrate is consumed, the rate continuously slows. To measure the initial rate, enzyme assays are carried out while the reaction has progressed only a few percent.
In our case, initial rates were measured for a range of substrate concentrations (0 mM - 9.0 mM NAM-NAG). The enzyme was then assayed over the same concentration range, but in the presence of 2 mM inhibitor X and 5 mM inhibitor Y. The data are available as a csv file.

Open the file in WordPad.
The first column shows the substrate concentrations.
The next columns show the initial reaction rates in the three conditions that have been tested.
To analyze the data we need to plot the initial rate of the reaction in function of the substrate concentration.

Which table type are you going to use ?
Plotting a variable that you have measured in function of a variable that you have manipulated is typical for an XY table.

Import the data.
Open the New Data Table (+ Graph) window Choose to create a XY table Import the data

Create the plot.
Click the corresponding graph in the Project Explorer Create the default Points only scatter plot

The initial rate of the reaction (v₀) increases as the substrate concentration (S) increases. However, as the substrate concentration gets higher, the enzyme becomes saturated with substrate and the rate reaches a maximum (Vmax). So the relation between V0 and S is not linear.

The Michaelis Menten equation models the non-linear relation between v₀ and S:

Prism allows use to use this equation for fitting a non-linear curve to our data and to estimate Vmax and K_M.

Fit a Michaelis-Menten model to the data.
Click the Analyze button in the Analysis section of the top toolbar: In the XY analyses section of the Analyze Data window select Nonlinear regression (curve fit) Expand the Enzyme kinetics - Substrate versus Velocity section Select Michaelis-Menten Click OK to see the curves superimposed on the graph:

What are Vmax and KM for the three conditions ?
In the Results section of the Project Explorer click the table called Nonlin fit of Kinetics