Question

I need to calculate the turnover rate of an enzyme using a lineweaver-burk plot. Is there a specific equation that should be used to calculate Kcat? I have been unable to find a straightforward answer on how to calculate Kcat!

Answer 1

• Michaelis - Menten equation is  
• Taking the reciprocal of this equation we get 1 / V0  = Km / Vmax * [S] + 1 / Vmax
• Thus Lineweaver - Burk equation =

  

• A plot of 1 / V0  with 1 /[S] gives a straight line ; such a plot is called double reciprocal / Lineweaver Burk plot.
• The maximal rate Vmax, gives the turnover number of an enzyme - which is defined as the number of substrate molecules converted into product by an enzyme molecule in a unit time when the enzyme is fully saturated with substrate.Denoted by kcat.
• kcat = Vmax/[E]T (general equation).
• kcat usually ranges from 1 to 104 per second.

Using Lineweaver Burk plot ;also we can determine kcat ;

• From the general equation ; kcat = Vmax / [E]T ;we get Vmax.
• Vmax = kcat * [E]T
• Substituting in the value of Vmax in the general equation of Lineweaver Burk plot we can thus calculate kcat ;

1 / V0  = Km / Kcat*[E]T*[S] + 1 / Kcat * [E]T

  On rearranging we get;

      1 / V0 = 1 / kcat * [E]T {Km+ [S] / [S] }

Thus Kcat calculated after again rearranging as follows

1 / kcat = 1 / V0 * [E]T { Km + [S] / [S] }