A tutorial of linear regression for estimating Vmax and Km 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 Velocity (mM/second)
concentrations (mM)
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 Vmax and Km of the Michaelis-Menten equation:

[S ]
V = Vmax , where V is the velocity of enzymatic reaction and [S] is the concentration
Km + [S ]
of the substrate.

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 ] Km [S ] K 1 1
= = + = m ⋅ +
V Vmax [ S ] Vmax [ S ] Vmax [ S ] Vmax [ S ] Vmax

1 Km 1 1
= ⋅ +
V Vmax [ S ] Vmax

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/Vmax (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/Km (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/Km).

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.

1. Open Excel 2007 and include the data into two columns:
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 R2 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 Vmax value. The Vmax value is the reciprocal of the
intercept on the y-axis (which is equal to my m-value). The m-value is 11.791
1 1
( = 11.791 ) and we calculate the Vmax: = V = 0.0847 . This means that my
V 11.791
Vmax value is 0.0847 mM/s.
10. We will now determine the Km 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
y = kx + m and set y=0 ( 0 = kx + m ) and solve this equation for x:
m 11.791 −1
x=− =− = −0.156 . By inverting this value ( = K m ) I have calculated
k 75.46 −0.156
the Km value to 6.39 mM.
You will now continue the lab by using the Km and Vmax values you have obtained by using
them for a mathematical model (Se computer lab 2).