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

Last updated

Jul 17, 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 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: Determine the type of inhibitors for Kinetics Experiments

Concerns: If you are low on Enzymes volumes, you can decrease your enzyme volume by half or by a 3rd.

Calculate the volume (in µL) needed to add the following amounts of Lauric Acid (Substrates) to your cuvettes:
- x16 → ______ µL (Optional)
- x8 → ______ µL
- x4 → ______ µL
- x2 → ______ µL
- x1 → (This is the volume of your Km)
- ÷2 → ______ µL
- ÷4 → ______ µL
- ÷8 → ______ µL
- ÷16 → ______ µL (Optional)
Set up seven (7) cuvettes (or more if you want to do the additional optional steps).
Without inhibitor: Follow the protocol from Activities 1-4. However, you will add Enzymes last, instead of NADPH
- You will use the Optimal enzyme concentration volume from Activities 3.1.
- You will be adjusting the volume of substrates and buffer for each cuvette tested.
- Note: If you are low in enzymes, cut the enzyme volume in half
With Inhibitors: Follow the same protocol from Step 3.
- You will use the Ki inhibitor volume from Activities 3.2.
- You will be adjusting the volume of substrates and buffer for each cuvette tested.
- Note: If you are low in enzymes, cut the enzyme volume in half
Determine the Velocity
Determine the inhibition percentages at each substrate concentration.
- Percent inhibition = 100 x (V_noninhibited - V_inhibited)/V_noninhibited
Plot Percent inhibition vs. Substrate concentration to a line 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 (Optional)	100	x16	previous result	IC₅₀		500
2	100	x8	previous result	IC₅₀		500
3	100	x4	previous result	IC₅₀		500
4	100	x2	previous result	IC₅₀		500
5	100	x1 (previous result)	previous result	IC₅₀		500
6	100	÷2	previous result	IC₅₀		500
7	100	÷4	previous result	IC₅₀		500
8	100	÷8	previous result	IC₅₀		500
9 (Optional)	100	÷16	previous result	IC₅₀		500

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.