This equation fits exactly the same curve as the equation that fits the turnover number Kcat rather than the Vmax.See the list of assumptions of all analyses of enzyme kinetics.Click and drag this table onto the Lineweaver-Burk graph, then click "Add" Notes Prism will generate a new data table titled "Plot a function". Enter Km/Vmax as the Slope (where Km and Vmax are the values reported by nonlinear regression earlier)ĩ. Calculate 1/Vmax and enter this value as the YIntercept (where Vmax is the value reported by nonlinear regression earlier)ħ. Switch to the "Parameter values & column titles" tabĦ. Use the "Range of X values" options at the bottom of the Function tab to specify where the line should start and endĥ. In the dialog that appears, select "Straight line" from the function list on the "Function" tabĤ. Select "Plot a function" from the "Generate curve" section of analyses, and click OKģ. From the graph of the transformed data, click the Analyze button in the Analysis section of the toolbarĢ. DO NOT simply perform linear regression, since this will not generate the correct line as mentioned above. Now, we'll want to add the appropriate line to the Lineweaver-Burk graph. Select the "Lineweaver-Burk" option and click OK (be sure that the "Create new graph of the results" checkbox is selected). In the "Function List" dropdown menu, select "Pharmacology and biochemistry transforms". 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. 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). Use nonlinear regression to obtain the most accurate values of Km and Vmax. The problem is that the transformations (reciprocals) distort the experimental error, so the double-reciprocal plot does not obey the assumptions of linear regression. If you do this, you won't get the most accurate values for Vmax and Km. Don't use the slope and intercept of a linear regression line to determine values for Vmax and Km. If you create a Lineweaver-Burk plot, use it only to display your data. One way to do this is with a Lineweaver-Burk plot, which plots the reciprocal of substrate concentration vs. Create a Lineweaver-Burk plotīefore nonlinear regression was available, investigators had to transform curved data into straight lines, so they could analyze with linear regression. It is the substrate concentration needed to achieve a half-maximum enzyme velocity. Km is the Michaelis-Menten constant, in the same units as X. 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. Vmax is the maximum enzyme velocity in the same units as Y. You can also choose Prism's sample data: Enzyme kinetics - Michaelis-Menten.Īfter entering data, click Analyze, choose nonlinear regression, choose the panel of enzyme kinetics equations, and choose Michaelis-Menten enzyme kinetics. If you have several experimental conditions, place the first into column A, the second into column B, etc. Enter substrate concentration into X, and enzyme velocity into Y. If your goal is to determine the turnover number kcat, rather than the Vmax, use an alternative version of the equation. The goal is to determine the enzyme's Km (substrate concentration that yield a half-maximal velocity) and Vmax (maximum velocity). The most common kind of enzyme kinetics experiment is to vary the concentration of substrate and measure enzyme velocity.
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