Question

Hi pls help! I need to plot the Michaelis-Menten curve for an enzyme in the presence of an inhibitor with the concentration 0.78 mM 2' Ado. The Vmax without inhibitor present is 1400 pM/s and the Km without inhibitor is 425 µM. I need to find Km and Vmax with inhibitor present to be able to plot it but I do not know how to find these with this information. Thank you!

Answer 1

Enzymes follow  Micahelis-Menten mechanism :

From Michaelis–Menten mechanism

rate (v) = k₂[E]₀/ 1 + Km/[S]₀

Micahelis-Menten mechanism equation can be rearranged to , this expression into a form that is easy to analyze data by linear regression:

1/v = 1/v_max + (Km/v_{max )} *1/[S]₀   (Lineweaver -burk plot equation )

Km = Micahelis-Menten constant ,Vmax : max. rate of reaction.

Lineweaver–Burk plot is a plot of 1/v against 1/[S]₀, and according to eqn , it will yield a straight line with slope of K_M/v_max, a y-intercept at 1/v_max, and an x-intercept at −1/K_M.

Slope and intercept in presence of Inhibitor depends on types of inhibition :

You have to plot your substrate conc. data and to get slope and intercept, you can use same Vmax and Km for caluclations ( as these are enzyme substrate specific), and equations have be derived in way to account for it.

• Competitive inhibitior , slope of the Lineweaver–Burk plot increases relative to the slope for data on the uninhibited enzyme and y-intercept does not change as a result of competitive inhibition .   slope :     Km (1+ [I] /K_I)/ Vmax
• In uncompetitive inhibition the inhibitor binds to a site of the enzyme that is remote from the active site, but only if the substrate is already present. The inhibition occurs because ESI reduces the concentration of ES.     y-intercept  = ( 1+ [I] /K_I ) / Vmax     ;   the slope does not change.
• Non-competitve inhibition : inhibitor binds to a site other than the active site, and its presence reduces the ability of the substrate to bind to the active site. Both the slope and y-intercept of the Lineweaver–Burk plot increase upon addition of the inhibitor.  

             y-intercept  = ( 1+ [I] /K_I ) / Vmax

              slope :     Km (1+ [I] /K_I)/ Vmax