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12: Activity 3-3 - Identifying the Type of Enzyme Inhibition

Last updated

Apr 19, 2025
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- 11: Activity 3-2 - Determining the IC₅₀ of Inhibitor
- Back Matter

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Learning Objectives

Define and distinguish between competitive and noncompetitive enzyme inhibition.
Interpret the effect of substrate concentration on percent inhibition.
Analyze experimental data to determine the type of inhibition.
Use graph-based results to calculate the inhibition constant (K_i).

Definition: Term

Enzyme Inhibitor: A molecule that binds to an enzyme and decreases its activity.
Competitive Inhibition: Inhibitor competes with the substrate for the enzyme’s active site. It increases K_m but does not affect V_max.
Noncompetitive Inhibition: Inhibitor binds to an allosteric site (not the active site), decreasing V_max but usually not affecting K_m.
IC₅₀: The inhibitor concentration that causes 50% inhibition of enzyme activity.
K_i (Inhibition Constant): A measure of inhibitor binding affinity. Lower K_i= stronger inhibitor.
% Inhibition: A calculated value that shows how much the inhibitor reduces enzyme activity, based on slope comparisons.

Note

Review your IC₅₀ data from previous experiments.
Understand how a 2-fold serial dilution works and why it’s used to generate a range of substrate concentrations.
Be prepared to work quickly when adding enzyme to each cuvette to maintain consistent reaction timing.

Identifying the Type of Enzyme Inhibition (Competitive vs. Noncompetitive)

In this experiment, you will determine whether an inhibitor behaves as a competitive or noncompetitive inhibitor by analyzing how the percent inhibition changes with varying substrate concentrations. This activity builds on your previous work in which you estimated the IC₅₀, the inhibitor concentration that causes 50% inhibition of enzyme activity. To understand the difference between competitive and noncompetitive inhibition, recall the following:

Competitive Inhibition: The inhibitor competes with the substrate for the enzyme’s active site. At low substrate concentrations, the inhibitor significantly reduces enzyme activity. However, as substrate concentration increases, the substrate can outcompete the inhibitor, leading to a decrease in percent inhibition. On a graph of % inhibition vs. substrate concentration, this appears as a downward-sloping line. The maximum reaction velocity (V_max) remains unchanged, but the apparent affinity for the substrate (K_m) increases.
Noncompetitive Inhibition: The inhibitor binds to a site other than the active site, and it can bind whether or not the substrate is bound. This results in a constant percent inhibition, regardless of substrate concentration, because increasing the substrate does not displace the inhibitor. The graph shows a horizontal line, indicating that the inhibitor affects V_max (lowers it) and usually does not affect K_m.

In this experiment, you will graph % inhibition vs. substrate concentration using your experimental data. Based on the shape of the curve:

A flat line suggests noncompetitive inhibition.
A declining line suggests competitive inhibition.

By comparing your curve to these expected patterns, you can identify the inhibition type and then calculate the inhibition constant (K_i). If you do not have access to your previous lab data, you may substitute in data from your group or another team.

Materials

100 mM potassium phosphate buffer, pH 7.4 OR 0.5X PBS
10 mM lauric acid in 50 mM potassium carbonate (Carolina #871840)
10 mM sodium myristate in 50 mM potassium carbonate (Sigma Aldrich #M3128-100G)
10 mM NADPH (Fisher Scientific #481973100MG)
100 mM imidazole in 100 mM potassium phosphate OR 0.5X PBS (Fisher Scientific #A1022122)
100 µM 4-phenylimidazole in 100 mM potassium phosphate OR 0.5X PBS (Fisher Scientific #L0223704)
Purified P450BM3 protein (from previous experiments)
UV/Visible Spectrophotometer (Thermo Scientific #840-300000)
UVette Cuvettes (Eppendorf #952010051)
Pipettes and pipette tips
Parafilm

Procedure

Use the same inhibitor volume and enzyme volume you used in your previous experiments to maintain consistency.
Label 10 cuvettes from #1 to #10.
- In Cuvette #1, add 32X the normal volume of substrate you typically use.
  - Example: If your standard substrate volume is ~15.6 µL, then 32× would be 500 µL.
    - ⚠️ IMPORTANT: The volumes used in this example are just for reference. Use the substrate volume from your own experiment! The principle is the same, but the numbers may vary slightly based on your actual 32X substrate volume.
Now, prepare your serial dilutions. (These volumes are based on the example above, your volume will be different!):
- Add 250 µL of potassium phosphate buffer (extraction buffer) to Cuvettes #2 through #9.
  - ⚠️ IMPORTANT: 250 µL is exactly half the volume of what's in Cuvette #1 — this is what allows you to make a 2-fold dilution!
- Transfer 250 µL from Cuvette #1 to #2, mix thoroughly. Cuvette #2 is now 16X.
- Transfer 250 µL from Cuvette #2 to #3, mix.
- Continue this process up to Cuvette #9.
- After mixing Cuvette #9, discard 250 µL to maintain equal volumes.
To each cuvette #1 to #9, add:
- The volume of inhibitor corresponding to the IC₅₀ from your earlier experiment
- 100 µL of 10 mM NADPH
- Potassium phosphate buffer to bring the total volume to 1 mL, excluding the enzyme
Add 1 mL of potassium phosphate buffer only to Cuvette #10 (Blank)
Gently mix all cuvettes (#1 to #9) and incubate at 37°C for at least 10 minutes. This allows the inhibitor and substrate to equilibrate.
Use Cuvette #10 to blank the spectrophotometer.
Quickly add the enzyme to cuvette #1. Cover with Parafilm, gently invert to mix.
- Immediately place it in the spectrophotometer and start the absorbance reading every 10 seconds for 2 minutes.
Repeat the process for each remaining cuvette (#2 to #9), working as quickly and consistently as possible.
Calculate % inhibition using the formula:
- Percent Inhibition=1−(Slope with Inhibitor/Slope without Inhibitor)×100
Create a Scatter Plot of % inhibition (Y-axis) against substrate concentration [S] (X-axis).
- Analyze your graph:
  - A flat line suggests noncompetitive inhibition.
  - A downward slope suggests competitive inhibition.

Cuvette #	10mM NADPH (uL)	Lauric Acid substrates (uL)	Enzyme CFE (uL)	Inmidazole (mM) 4-phenylimidazole (uM)	PO₄ buffer (uL)	Total Volume (uL)
1	100	x16	previous result	IC₅₀		1000
2	100	x8	previous result	IC₅₀		1000
3	100	x4	previous result	IC₅₀		1000
4	100	x2	previous result	IC₅₀		1000
5	100	x1 (previous result)	previous result	IC₅₀		1000
6	100	÷2	previous result	IC₅₀		1000
7	100	÷4	previous result	IC₅₀		1000
8	100	÷8	previous result	IC₅₀		1000
9	100	÷16	previous result	IC₅₀		1000
10 (Blank)	0	0	0	0	1000	1000

Interpreting the Sample Graph

In the sample graph shown, two sets of data are plotted:

The X symbols represent competitive inhibition. The line slopes downward as substrate concentration increases, indicating that more substrate overcomes inhibition.
The O symbols represent noncompetitive inhibition. The line is flat, showing that substrate concentration has no effect on the degree of inhibition.

This graph provides a visual key for interpreting your own results.

Reflective Questions

What does the shape of your % inhibition vs. substrate concentration graph tell you about your inhibitor?
If the line is flat, what does that mean for substrate competition?
If the line slopes downward, how does that relate to the enzyme’s active site?
How did your experimental values compare to your theoretical understanding of inhibition?
What could affect the accuracy of your K_i value?
Now that you’ve identified the inhibition type:
- Use the appropriate equation to calculate K_i:
  - For competitive inhibition: K_i=IC₅₀/(1+([S]/K_m)
  - For noncompetitive inhibition: K_i≈IC₅₀
Reflect on what the K_i tells you about the inhibitor’s strength and potential usefulness in a therapeutic or experimental context.