Topic #5: Enzyme Inhibition Calculations
Recommended problems in Campbell, Chapt. 5: #25, 26.
Model reaction: EI <=>
E + S <=> ES > E + P
 The MichaelisMenten equation with a competitive inhibitor present:

v_{o} = V_{max}[S]/(aK_{M} + [S] )
, where
 v_{o} = the initial velocity;
 V_{max} = the maximal velocity;
 K_{M} = the Michaelis constant;
 a = (1 + [ I ]/K_{I}), and K_{I} is the dissociation constant of the inhibitor, I.
We want to determine the value of K_{I} and compare it to K_{M} (or to the K_{I} 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.
(Each calculated v_{o} value has a small "experimental error" added to it.)
3. Record the values of [S] and v_{o} you obtain with [ I ] = 0; then graph them on a double reciprocal plot to determine K_{M} and V_{max}.
4. Next, set [S] = K_{M} and vary [ I ] to find a concentration that decreases v_{o} to about 3040% of V_{max}. (This will be roughly the K_{I} 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 K_{I} 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.
