BL4820 BASIC BIOCHEMICAL TECHNIQUES -- Lecture 3 - Enzyme Kinetics -- Expt 2 Part B
3. Calculating Km and Vmax from Experimental Data
The best solutions to the problem of calculating Km and Vmax are to use a computer program. I have a computer program available in my lab that runs on a PC (called EnzPack) which you can use to obtain these kinetic constants for your data (It also has a tutorial on enzyme kinetics). But even if you use the computer, you must also make a graphical representation of these results using the "Lineweaver-Burk" plot, which is a linear transformation of the Michaelis-Menton equation generated by taking the reciprocal of both sides of the equation:
1/vo = 1/Vmax + ((Km/Vmax) * (1/[S]))
Figure 4. Equation for Lineweaver-Burk Plot (Linear Transformation of M&M Equation).
To use this equation and make the graph required for your lab report, you take the reciprocal of each of your datum points or in other words for each v you calculate 1/v and for each [S] you calculate 1/[S], then you plot 1/v versus 1/[S]:
Graph 2. Double Reciprocal Plot of 1/v versus 1/[S].
You get the Km and Vmax from the graph as shown on it. Since the above equation is the one for a line (i.e. y = a + bx), you can make a linear regression or "least-squares fit" of your data for this graph and calculate Km and Vmax from the equation of the line.