Spreadsheet Activity #12b: Enzyme Kinetics - Lineweaver-Burke & Schatchard Data Fits
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Recall from Spreadsheet Activity #12a, that the rates of enzyme-catalyzed reactions are dependent on the amount of catalyst, the concentration of substrate (reactant), the affinity of the substrate for the enzyme, and the maximum velocity at which the enzyme can function. The "velocity" of an enzyme can be accurately portrayed by the Michaelis-Menten equation:
V = Vmax * [S] / ([S] + Km)
where V = velocity, Vmax = maximum velocity, [S] = substrate concentration, and Km is the Michaelis-Menton constant. By plotting [S] on the x-axis and V on the y-axis, a hyperbolic curve is obtained. However, this form of the equation is difficult to use when determining values for Km and Vmax.
Alternatively, the reciprocal of both sides yields a linear plot as first described by Lineweaver and Burke. Unlike the Michaelis-Menton plot, the Lineweaver-Burke plot of the data has no asymptotes, making it easier to find the values for Vmax and substrate concentration. Through a few steps not shown here, the above equation can be rearranged to:
1/V = (1/Vmax) + (Km/Vmax) * (1/[S])
This form of this equation is linear .The data is in the form of y = b + mx. Create another table below the first with a column for 1/[S] and a column for 1/V. The X intercept is equal to –1/Km and the Y intercept is 1/Vmax with the slope being Km/Vmax. Calculate and report Vmax and Km.
Another linearized form of the Michaelis-Menton plot is the Scatchard Plot. In this plot, V/[S] is plotted on the x-axis and V is plotted on the y-axis. This yields a linear plot with a negative slope. Vmax is read directy from the y-intercept and Km is the negative slope. Create a Scatchard Plot and report values for Km and Vmax from this chart.
(Click to download data: 12bEnzKin)
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Spreadsheet 12: Enzyme
Kinetics - Lineweaver-Burke
& Schatchard Data Fits |
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Actual Data: |
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[S] |
V |
1/[S] |
1/V |
V/[S] |
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(umol/L) |
(nmol/L/min) |
. |
. |
. |
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0.00 |
0.0 |
. |
. |
. |
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8.33 |
13.8 |
. |
. |
. |
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10.00 |
16.0 |
. |
. |
. |
|
12.50 |
19.0 |
. |
. |
. |
|
16.70 |
23.6 |
. |
. |
. |
|
20.00 |
26.7 |
. |
. |
. |
|
25.00 |
30.8 |
. |
. |
. |
|
33.30 |
36.3 |
. |
. |
. |
|
40.00 |
40.0 |
. |
. |
. |
|
50.00 |
44.4 |
. |
. |
. |
|
60.00 |
48.0 |
. |
. |
. |
|
80.00 |
53.4 |
. |
. |
. |
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100.00 |
57.1 |
. |
. |
. |
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200.00 |
66.7 |
. |
. |
. |
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Lineweaver-Burke |
y-Intercept |
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Km = |
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x-Intercept |
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Vmax = |
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Scatchard |
y-Intercept |
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Km = |
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x-Intercept |
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Vmax = |
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