Competitive Inhibition

1. Estimate KI for a competitive inhibitor when [I] = 5 mM gives an apparent value of Km that is
four times the Km for the uninhibited reaction.

Solution: From the equation

Vmax [S ] Vmax [S ]
v 
 I    [S ]
K m,app
K m 1  [S ]

 K I 

Kmapp = 4 Km

4Km = Km(1+(5x10-3/KI)

4 = 1 + 5x10-3/KI

3 = 5x10-3/KI

KI = 1.67x10-3 M

Source: https://quizlet.com/7607446/biochemistry-ch-12-problems-flash-cards/

2. The following data were obtained for a competitive inhibition study in which the [I] = 3 uM
for each determination of vo in the presence of inhibitor.

Vmax = 200 uM P/min for both data sets.

a) Determine Km for the data obtained in the absence of inhibitor.
b) Determine Km, app for the data obtained in presence of inhibitor.
c) Calculate the value for Ki.

Solution:
Competitive Inhibition

a) Determine Km for the data obtained in the absence of inhibitor.

Since Vmax= 200, Vmax/2 = 100

Km = [S] that gives Vo = Vmax/2,

so Km = 10 uM

b) Determine Km, app for the data obtained in presence of inhibitor.

At Vo =100, Km, app = 30 uM

c) Calculate the value for Ki.

Km, app = Km (1+ [I]/Ki)

30 uM = 10 uM(1 + 3 uM/Ki )

3 = 1 + (3 uM/Ki)

2 = 3 uM/Ki

Ki = 3 uM/2 = 1.5 uM

Source:
http://cbc.arizona.edu/classes/bioc460/summer/460web/lecture/EnzInhibProblems.pdf
Competitive Inhibition

3. Lactose is a disaccharide found in milk. Although in the United States we are told that "milk,
it does a body good," many adults throughout the world get sick from drinking milk because
they cannot digest lactose. Lactose intolerance varies markedly among various human
populations. (For example, only about 3% of people of Danish descent are lactose intolerant,
compared with 97% of people of Thai descent.) When someone who is lactose intolerant ingests
milk, the lactose accumulates in the lumen of the small intestine because there is no mechanism
for uptake of the disaccharide. This causes abdominal distension, cramping, and watery
diarrhea.

You decide to study lactose further, and see whether it can also cleave other common
disaccharides, such as maltose. (Maltose = glucose + glucose.) You find that maltose is NOT
cleaved by lactase, and furthermore, maltose appears to have some kind of inhibitory effect on
lactase's ability to cleave lactose.

You quantitatively study the kinetics of lactase with lactose alone, and in the presence of both
lactose and maltose. You measure the initial velocity of the reaction (rate at which lactose is
cleaved) at varying concentrations of substrate. The data is given below.

[Lactose] moles/liter Velocity (moles/min)

lactose only with maltose
0.3 x 10-5 10.4 4.1
0.5 x 10-5 14.5 6.4
1.0 x 10-5 22.5 11.3
3.0 x 10-5 33.8 22.6
9.0 x 10-5 40.5 33

Make a Lineweaver-Burke plot for lactase both with and without maltose. Determine the type
of inhibition.

Solution:

Using the Lineweaver-Burke plot for Michaelis-Menten Kinetics,

1 Km 1 1
 
v Vmax [S ] Vmax
Plot 1/V (y) vs. 1/[S] (x) for both data
Competitive Inhibition

0.3

0.25 y = 7E-07x + 0.0225

R² = 1

0.2
Axis Title

Without Inhibitor
0.15
With Inhibitor
Linear (Without Inhibitor)
0.1 y = 2E-07x + 0.0224
R² = 0.9988 Linear (With Inhibitor)

0.05

0
0 50000 100000 150000 200000 250000 300000 350000
Axis Title

Lactose only:

Vmax = 1/0.0224 = 44.6429 mol/min

Km = (1/0.0224)(2x10-7) = 1x10-5 mol/L

With Lactose:

Vmax = 1/0.0225 = 44.4444 mol/min

Km,app = (1/0.0225)(7x10-7) = 2.96x10-5 mol/L

Competitive Inhibition

Source: http://dwb4.unl.edu/chem/chem869k/chem869klinks/esg-
www.mit.edu/esgbio/eb/solutions.html
Competitive Inhibition

4. You have performed a series of experiments determining the Ki values for three competitive
inhibitors. The following table lists the results:

Inhibitor K i (uM)

A 5
B 1
C 0.2

a) Which inhibitor binds with higher affinity to the free enzyme?

The inhibitor with the lowest Ki (a dissociation constant) binds with the highest affinity, so the answer
is inhibitor C.

b) If the same concentration of inhibitor were used in each experiment, which inhibitor would
give the smallest value of Km,app?
The smallest Km,app value implies the weakest binding of I, or the least amount of
competition for E. Inhibitor A would give the lowest value for Km,app.

c) If the value for Km is 1 uM, what is the ratio of Ki/Km for each inhibitor? How is this related to the
competing equilibria for binding of the substrate vs. the inhibitor to the enzyme?
The ratio of Ki/Km directly compares the binding constants for both the inhibitor and the substrate.
If Ki/Km > 1, the substrate is bound tighter, so inhibitor will not have that much effect on kinetics.
If Ki/Km = 1, both substrate and inhibitor bind with same affinity.
If Ki/Km < 1, the inhibitor binds with greater affinity, therefore will have a “stronger” effect
on the kinetics.
In this case, Ki/Km = 5, 1, and 0.2 for A, B, and C, respectively. Thus, as was the case in (a), C binds with
greatest affinity and will have a greater effect on the enzyme kinetics than A or B.