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A tutorial of linear regression for estimating V max and K m values with Excel

By Andreas Jansson

In the laboratory you should have measured the velocity for a number of different substrate concentrations. I will use an example from where they have the following data:

Table 1: Collected data

 Substrate concentrations (mM) Velocity (mM/second) 2.5 0.024 5.0 0.036 10.0 0.053 15.0 0.06 20.0 0.064

With these data we want to find the values of V max and K m of the Michaelis-Menten equation:

V

=

V

max

[

S

]

K

m

+ [

S

]

of the substrate.

, where V is the velocity of enzymatic reaction and [S] is the concentration

If we plot this function (the enzymatic velocity) as a function of the substrate concentration we will get the following figure:

Figure 1. The reaction velocity, v, as a function of the substrate concentration, [S], for an enzyme. This relationship is called the Michaelis-Menten equation.

Since this curve is not linear it is difficult to fit a line to our data (it is of course possible but then we need to learn about nonlinear regression which is quite difficult). It would be nice if we could some how convert the equation so that we get a straight line since it will be a lot

easier to fit a straight line to our data (which is called linear regression). The Lineweaver- Burk equation is used to make the Michaelis-Menten equation linear by taking its reciprocal:

 1 = K m + [ S ] = K m + [ S ] = K m ◊ 1 + 1 V V max [ S ] V max [ S ] V max [ S ] V max [ S ] V max
 1 = K m ◊ 1 + 1 V V max [ S ] V max

By using the Lineweaver-Burk equation we will be able to make a Lineweaver-Burk plot which is linear and where the intercept on the y-axis gives us: 1/V max (Set [S]=0 in the above equation (intercept on the y-axis) and you will see that 1/V=1/Vmax), and the intercept on the x-axis gives us -1/K m (set 1/V=0 in the above aquation (intercept on the x-axis) and solve for 1/[S] and you will see that 1/[S]=-1/K m ).

Figure 2. Lineweaver-Burk plot for an enzyme that shows upon Michaelis-Menten kinetics.

Note that the axes in figure 2 are the reciprocals, which means that we fist have to take the reciprocals of our data (Table 2).

Table 2: Reciprocal of collected data from Table 1

 1/[S] 1/V 0,4 41,667 0,2 27,778 0,1 18,868 0,067 16,667 0,05 15,625

We will now include the values from Table 2 into Excel.

2. Select the two columns with the data. Click Insert -> Select the scatter icon -> select scatter with only markers (top-left icon “on my computer”).

3. You should now have got a figure with the data points. We will now change the names on the axes.

4. Click the “Design tab” (probably already selected) and then select “Layout 1” in the “Chart Layouts” box. You should now have got axes titles in your figure. Rename these titles by just clicking on them and type: “1/[S]” for the x-axis and “1/V” for the y-axis.

5. We will now add a linear regression line to our data. Make sure that the figure is selected and click on the tab called “Layout”. Select the icon called “Trendline” and the option “Linear Trendline”. You should now have got a linear line in your figure.

6. We will now expand this line so that it intercepts the x and y axes. First, “right-click” on the line in your figure. A window should now pop-up, in which you select the option “Format Trendline…”. The following window should now appear on your screen:

7.

In the forecast box, you should increase “Backward from 0,0 to 0,3”.

8. Also, tick the box “Display Equation on chart” and the box “Display R-squared value on chart”. Close this box. You should now have the following figure:

The equation for the linear regression is y = kx + m where k is the slope of the line (you can see that my slope is 75,46) and m represents the intercept of the y-axis (my m-value is 11.791). The R 2 value is the square of the correlation coefficient which gives us a measure of the reliability of the linear relationship between the x and y values (values close to 1 indicate excellent linear reliability).

9. We should now determine the V max value. The V max value is the reciprocal of the intercept on the y-axis (which is equal to my m-value). The m-value is 11.791

0.0847 . This means that my

V max value is 0.0847 mM/s. 10. We will now determine the K m value. We can see that the line intercept the x-axis at about -0.18. However, we can calculate this value more precisely by using the formula

 ( 1 = 11.791 ) and we calculate the V max : V

1

11.791 = V =

y = kx + m

x =

m

=

k

and

11.791

=

75.46

set

y=0 ( 0 = kx + m ) and solve this equation for x:

) I have calculated

1

0.156

0.156

. By inverting this value (

= K

m

You will now continue the lab by using the K m and V max values you have obtained by using them for a mathematical model (Se computer lab 2).