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9: Activity 3-0 - Project Instructions - Investigating Enzyme Kinetics

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Background: Molecular Analysis of P450 Enzyme Reaction Rates

Enzymes are biological catalysts that accelerate chemical reactions. One of the most important ways to understand how enzymes work is by studying their kinetics—how fast they convert substrates into products under different conditions. A few key terms will guide your understanding of enzyme kinetics:

V_max is the maximum rate of a reaction when the enzyme is saturated with substrate.
K_m (Michaelis-Menten constant) is the substrate concentration at which the reaction rate is half of Vmax. It provides insight into the enzyme’s affinity for the substrate—a lower Km means higher affinity.
K_cat (turnover number) is the number of substrate molecules an enzyme can convert into product per unit time when fully saturated.

In this project, you will explore the enzyme Cytochrome P450_BM3, which plays an important role in fatty acid metabolism. In the presence of NADPH and oxygen, P450_BM3 hydroxylates long-chain fatty acids like lauric acid. It uses several cofactors—FAD, FMN, and heme—to transfer electrons from NADPH to the enzyme’s active site. The enzyme follows a classic reaction mechanism: E+S ⇌ ES → E+P, where E is the enzyme, S is the substrate, ES is the enzyme-substrate complex, and P is the product. The rate of the reaction depends on how much enzyme and substrate are available. Early in the reaction, when substrate is abundant and product levels are low, we can focus on the initial reaction rate—the steepest, linear portion of the product vs. time graph.

As the substrate concentration increases, so does the rate—until the enzyme becomes saturated. At this point, further increases in substrate no longer affect the rate. This behavior is described by the Michaelis-Menten equation: v = V_max⁡[S] / K_m+ [S]

This equation helps us estimate kinetic parameters from experiments. However, determining V_max from a graph is tricky since it requires an impractically high amount of substrate. A more convenient way to estimate kinetic values is to use the Lineweaver-Burk plot, a double reciprocal plot that provides a straight-line graph where V_max and K_m can be directly determined from the y- and x-intercepts.

In this project, you will analyze the activity of P450_BM3 by exploring how substrate concentration, enzyme concentration, and inhibitors affect the enzyme’s rate of reaction. You will also examine whether certain compounds act as competitive or non-competitive inhibitors.

Project Title: Molecular Analysis of P450 Enzyme Kinetics

Objective:

You will work in a group of 3–4 students to investigate the enzyme activity of Cytochrome P450_BM3, focusing on reaction kinetics and the effect of inhibitors (Imidazole and 4-Phenylimidazole) using lauric acid as the substrate. Your goal is to analyze whether these compounds act as competitive or non-competitive inhibitors and present your findings in the form of a scientific research poster.

Timeline:

This is a four-day project. You will plan, perform, and analyze your experiments, culminating in an oral poster presentation of your findings.

Overview:

You will apply multiple laboratory techniques discussed in class, including:

Protein quantification using the Bradford assay
Absorbance measurements using Beer’s Law
Enzyme kinetics analysis

Project Flow:

Form a Group of 3–4 students.
Select a Research Question:
- Option 1: Does Imidazole act as a competitive or non-competitive inhibitor of P450_BM3?
- Option 2: Does 4-Phenylimidazole act as a competitive or non-competitive inhibitor of P450_BM3_?
Create a flowchart of your experiment.
- Define your experimental variables and controls.
- Decide how you will measure the initial rate of reaction.
Perform the Experiment:
- Collect data using proper lab techniques.
- Measure product formation over time.
- Use graphs to determine initial velocities, Vmax, and Km.
Data Analysis:
- Use both Michaelis-Menten and Lineweaver-Burk plots.
- Compare rates with and without inhibitors to determine the type of inhibition.
- Interpret your data in the context of enzyme-substrate interaction.
Poster Presentation:
- Prepare a clear and engaging scientific poster.
- Include sections on background, methods, results (graphs/tables), and conclusions.
- Present your poster to the class and explain how your data supports your research question.

What to Include in Your Poster

Your poster must contain the following sections with organized, visually clear, and well-labeled figures and tables:

Title and Group Info

A clear, specific project title
Names of group members
School name and sponsorships

Background

Explain what Cytochrome P450_BM3does and why it’s important in research, clinic, and application
Describe what K_m, V_max, and K_cat mean and why is it being studied in your project
Introduce enzyme inhibition: competitive vs. non-competitive (with a simple diagram if you like)
Mention your specific inhibitors (Imidazole, 4-Phenylimidazole) and hypothesis

Objective

Clearly state your research question

Methods

Use a flowchart or bullet list to summarize the experimental steps:
- Highlight your control groups and replicate strategy

Results

Include the following results using figures, tables, and brief captions:

Figure 1. Protein Quantification and Beer’s Law Measurements

Figure 1a - Bradford standard curve (Scatter plot graph: absorbance vs. protein concentration)
Figure 1b - Table comparing protein concentration and Beer's Law concentration
- Calculated enzyme concentration in your samples
- Conversion of absorbance from Beer's Law to concentration (Table)

Figure 2. Troubleshooting Concentration

Figure 2a - Optimal Enzyme Concentration versus time (Line graph showing different steep curves)
Figure 2b - Michaelis-Menten Plot: V₀ vs. [S], with V_max and K_m estimated (Scatter plot graph)
- Indicate the best optimal substrate concentration used for kinetic inhibitor study.

Figure 3. Kinetic Analysis

Clearly show comparison between control and inhibitor conditions

Figure 3a - Containing noninhibited and inhibited curves in the Michaelis-Menten Plot: V₀ vs. [S], with V_max and K_m estimated (Scatter plot graph)
Figure 3b - Lineweaver-Burk Plot: 1/V₀ vs. 1/[S] to confirm inhibition type (Scatter plot graph)
Figure 3c - Include a table:
- V_maxand K_m (for control and inhibitor)
- Interpretation of inhibition (competitive/non-competitive)

Conclusion

What type of inhibition did each drug show?
Did the results support your hypothesis?
What did you learn about enzyme kinetics?
What were sources of error or limitations?
How could the experiment be improved?

References

Include any scientific papers, lab manuals, or sources you used (at least 2–3 references, APA or MLA format).

Poster Tips

Keep text clear and concise
Use large fonts and simple language
Label all graphs and figures with titles and axes
Use color coding or visuals to compare control vs. inhibitor groups
Make sure your story is logical and easy to follow—background ➝ hypothesis ➝ methods ➝ data ➝ interpretation

Presentation Expectations

At the end of the project, each group will present their poster to the class. Be prepared to:
- Explain your results and conclusions
- Answer questions from peers and instructors
- Speak for about 4–8 minutes (~up to 2 minutes per student)
  - Every group member must speak

Grading Rubric – Total: 100 points

1. Experimental Results & Data Analysis – 40 points

All required results are clearly presented using graphs, tables, and labeled figures (Michaelis-Menten plots, Lineweaver-Burk plots, standard curves, kinetics, protein concentration).
Accurate interpretation of kinetic data, including Vmax, Km, and inhibitor impact.
Provides correct identification of inhibition type (competitive vs. noncompetitive) based on evidence.
Quantitative and/or statistical analysis is present and appropriate.
Visuals are properly labeled, include units, and support the group’s conclusions.

2. Q&A and Understanding of Concepts – 30 points

Group demonstrates a deep understanding of their data and experimental methods.
Students can clearly explain what their results mean and how they reached their conclusions.
Confidently and accurately answer instructor questions about:
Enzyme kinetics concepts (Vmax, Km, Kcat)
Types of inhibition
Experimental design and reasoning
Troubleshooting or limitations

3. Background & Introduction – 10 points

Objective and research question are clearly stated.
Provides a concise, accurate summary of P450BM3, the chosen inhibitors (Imidazole, 4-Phenylimidazole), and substrate (Lauric Acid).
Clearly explains the significance of determining inhibition type in biochemistry or biotechnology.

4. Methods (Flowchart Format) – 10 points

Presents a clear, logical flowchart of the experimental steps.
Includes major techniques: Bradford assay, Beer’s Law, enzyme kinetics, protein purification, etc.
Shows understanding of the purpose of each method in the overall workflow.

5. Presentation Quality & Visual Design – 10 points

Poster is visually organized, clean, and easy to follow.
Text is concise; fonts and colors enhance readability.
Group delivers a clear, engaging oral presentation—each member contributes.
Poster includes: Title, Introduction, Methods, Results, Discussion, Conclusion, and References.

Summary of Grading Criteria:

Category	Points
Experimental Results & Analysis	40
Q&A and Scientific Reasoning	30
Background & Introduction	10
Methods (Flowchart)	10
Visual Design & Oral Presentation	10
Total	100