GraphPad Prism 10 Curve Fitting Guide (2024)

Introduction

The most common kind of enzyme kinetics experiment is to vary the concentration of substrate and measure enzyme velocity. The goal is to determine the enzyme's Km (substrate concentration that yield a half-maximal velocity) and Vmax (maximum velocity). If your goal is to determine the turnover number kcat, rather than the Vmax, use an alternative version of the equation.

Step by step

Create an XY data table. Enter substrate concentration into X, and enzyme velocity into Y. If you have several experimental conditions, place the first into column A, the second into column B, etc.

You can also choose Prism's sample data: Enzyme kinetics -- Michaelis-Menten.

After entering data, click Analyze, choose nonlinear regression, choose the panel of enzyme kinetics equations, and choose Michaelis-Menten enzyme kinetics.

Model

Y=Vmax*X/(Km+X)

GraphPad Prism 10 Curve Fitting Guide (1)

Interpret the parameters

Vmax is the maximum enzyme velocity in the same units as Y. It is the velocity of the enzyme extrapolated to very high concentrations of substrate, so its value is almost always higher than any velocity measured in your experiment.

Km is the Michaelis-Menten constant, in the same units as X. It is the substrate concentration needed to achieve a half-maximum enzyme velocity.

Create a Lineweaver-Burk plot

Before nonlinear regression was available, investigators had to transform curved data into straight lines, so they could analyze with linear regression. One way to do this is with a Lineweaver-Burk plot, which plots the reciprocal of substrate concentration vs. the reciprocal of enzyme velocity.

GraphPad Prism 10 Curve Fitting Guide (2)

If you create a Lineweaver-Burk plot, use it only to display your data. Don't use the slope and intercept of a linear regression line to determine values for Vmax and Km. If you do this, you won't get the most accurate values for Vmax and Km. The problem is that the transformations (reciprocals) distort the experimental error, so the double-reciprocal plot does not obey the assumptions of linear regression. Use nonlinear regression to obtain the most accurate values of Km and Vmax.

To create a Lineweaver-Burk plot (with corresponding line), you'll first want to make note of the values that the nonlinear regression reported for Vmax and Km (we'll need these later). First, from the data table containing the data, click the "Analyze" button in the toolbar, select Transform from the "Transform, Normalize..." section of analyses, and click OK. In the "Function List" dropdown menu, select "Pharmacology and biochemistry transforms". Select the "Lineweaver-Burk" option and click OK (be sure that the "Create new graph of the results" checkbox is selected).

Now, we'll want to add the appropriate line to the Lineweaver-Burk graph. DO NOT simply perform linear regression, since this will not generate the correct line as mentioned above. Instead, follow these steps:

1.From the graph of the transformed data, click the Analyze button in the Analysis section of the toolbar

2.Select "Plot a function" from the "Generate curve" section of analyses, and click OK

3.In the dialog that appears, select "Straight line" from the function list on the "Function" tab

4.Use the "Range of X values" options at the bottom of the Function tab to specify where the line should start and end

5.Switch to the "Parameter values & column titles" tab

6.Calculate 1/Vmax and enter this value as the YIntercept (where Vmax is the value reported by nonlinear regression earlier)

7.Enter Km/Vmax as the Slope (where Km and Vmax are the values reported by nonlinear regression earlier)

8.Click OK

9.Prism will generate a new data table titled "Plot a function". Click and drag this table onto the Lineweaver-Burk graph, then click "Add"

Notes

See the list of assumptions of all analyses of enzyme kinetics.

This equation fits exactly the same curve as the equation that fits the turnover number Kcat rather than the Vmax. The product of Kcat times Et (the concentration of enzyme sites) equals the Vmax, so if you know Et, Prism can fit kcat.

This equation is a special case of the equation for allosteric enzymes. That allosteric model adds an additional parameter: the Hill slope h. When h equals 1.0, the two models are identical.

Note that Km is not a binding constant that measures the strength of binding between the enzyme and substrate. Its value takes into account the affinity of substrate for enzyme, and also the rate at which the substrate bound to the enzyme is converted to product.

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GraphPad Prism 10 Curve Fitting Guide (2024)
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