Biochemistry I Fall Term
Calculations & Graphing

Topic #5: Enzyme Inhibition Calculations

Recommended problems in Campbell, Chapt. 5: #25, 26.
Model reaction:     EI <=> E + S <=> ES --> E + P
The Michaelis-Menten equation with a competitive inhibitor present:
vo = Vmax[S]/(aKM + [S] ) ,  where
vo = the initial velocity;
Vmax = the maximal velocity;
KM = the Michaelis constant;
a = (1 + [ I ]/KI), and KI is the dissociation constant of the inhibitor, I.
We want to determine the value of KI and compare it to KM (or to the KI for other inhibitors). The general approach is to determine the dependence of the initial velocity on the substrate concentration at one or more fixed concentrations of inhibitor. (You should do the Enzyme Kinetics example before working this problem.) This is done in four steps.
1. Enter values for [S] and [ I ] in these boxes:
[S] = mM [ I ] = mM
2. For the above values of [S] and [I], Calculate vo
vo = mM/min  
    (Each calculated vo value has a small "experimental error" added to it.)

3. Record the values of [S] and vo you obtain with [ I ] = 0; then graph them on a double reciprocal plot to determine KM and Vmax.
4. Next, set [S] = KM and vary [ I ] to find a concentration that decreases vo to about 30-40% of Vmax. (This will be roughly the KI value.) Then keep [ I ] fixed at that concentration, and vary [S] to determine a complete substrate saturation curve.
Plot these data on the original double reciprocal plot. Consult the Lecture 15 notes for various ways of calculating KI from these data. (An example is also shown on the sample Answer Sheet, linked below.) It is good practice to determine substrate saturation curves at two or more concentrations of the inhibitor.
Answers to this problem.

A sample Answer Sheet for a similar problem shows the format of the results and the graph required.

Back to the Calculations & Graphing Index