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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-week 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
Data 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 (8 points)

A clear, specific project title
Names of group members
School name and sponsorships
Include any scientific papers, lab manuals, or sources you used (at least 2–3 references, APA or MLA format).
Overall looks and presented in a professional manner

Background/Objective (5 points)

(1pts) What were your research question(s) and the goal of your project?
(1pts) Why is Cytochrome P450_BM3important in research, clinics, and applications?
(1pts) What were the K_m and V_max for your substrate and inhibitor?
(1pts) What type of inhibitor was your drug?
(1pts) How would the type of inhibition be relevant or significant in clinical and real-world applications?

Methods (5 points)

(2pts) Explain the list of experiments you did via the use of a flowchart to summarize each test (I love cartoon images):
- (3pts) Choose three key experiments, and explain what were your control groups and why,

Results (39 points)

Figure 1. Protein Expression (From Project 3)
- (2pts) Figure 1a - Scatter plot from Bradford standard curve (Showing R²and equation)
  - (1pts) Explain what R² represents
  - (1pts) What was the concentration of your CFE (mg/mL)?
- (2pts) Figure 1b - Pie Chart from Beer's Law (Percent of your P450BM3 is in CFE)
  - (1pts) What was the concentration of your p450bm3?
Figure 2. Determining the Km for enzyme and substrate
- (2pts) Figure 2a - Line graph from kinetics of different Enzymatic concentrations (Enzyme concentration vs time)
  - (1pts) Which linear line do you choose and why?
  - (1pts) What was the concentration of the enzyme you used for the remaining experiment?
- (2pts) Figure 2b - Scatter plot graph (Michaelis-Menten) from kinetics of different substrate concentrations (Velocity vs. substrate)
  - (1pts) What were your estimated V_max and K_m?
  - (1pts) What was the concentration of the substrate you used for the remaining experiment?
- (2pts) Figure 2c - Scatter plot graph (Lineweaver-Burke) from the inverse of figure 2b (1/Velocity vs. 1/substrate)
  - (1pts) What were your calculated V_max and K_m?
  - (1pts) Explain the relationship between V_max and K_m, and what do these values mean?
Figure 3. Determining the Ki of the inhibitor
- (2pts) Figure 3a - Scatter plot graph (Michaelis-Menten) from kinetics of different inhibitor concentrations (Velocity vs. inhibitor)
- (2pts) Figure 3b - Scatter plot graph (Michaelis-Menten) from kinetics of different inhibitor concentrations (% inhibition vs. inhibitor)
  - (1pts) What were your estimated inhibitor V_max and Ki?
  - (1pts) What was the concentration of the inhibitor you used for the remaining experiment?
- (2pts) Figure 3c - Scatter plot graph (Lineweaver-Burke) from the inverse of Figure 3a (1/Velocity vs. 1/inhibitor)
  - (1pts) What were your calculated V_max and Ki?
  - (1pts) Compare and contrast K_i to K_m, and what do these values mean?

Figure 4. Identifying the type of Inhibition
- (2pts) Figure 4a - Scatter plot graph (Michaelis-Menten) from kinetics displaying inhibited vs noninhibited (Velocity vs. inhibitor)
- (2pts) Figure 4b - Scatter plot graph (Lineweaver-Burke) from the inverse of Figure 4a (1/Velocity vs. 1/inhibitor)
  - (2pts) Observe the relationship with the graphs, and explain the evidence showing whether your inhibitor is competitive vs non-competitive.
- (2pts) Figure 4c - Scatter plot graph (Relationship) from kinetics of displaying substrate vs inhibitor (% inhibition vs. substrates)
  - (2pts) Observe the relationship with the graphs, and explain the evidence showing whether your inhibitor is competitive vs non-competitive.

Discussion (3 points)

(2pts) What were the sources of error or limitations you ran into?
(1pts) What experiment could you improve?

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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 all questions
- The instructor can call on any student to answer his question.
  - Make sure that everyone in your team understands the project, not only their small part!

Grading Rubric – Total: 60 points

1. Results - Experimental Results – 22 points

All results are clearly presented using graphs, tables, and labeled figures (Michaelis-Menten plots, Lineweaver-Burk plots, standard curves, kinetics, protein concentration).
Quantitative and/or statistical analysis is present and appropriate.
Visuals are properly labeled, include units, and support the group’s conclusions.

2. Results - Q&A and Understanding of Concepts – 17 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

3. Background & Discussion – 8 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.

4. Methods (Flowchart Format) – 5 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 – 8 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	22
Q&A and Scientific Reasoning	17
Background & Introduction	8
Methods (Flowchart)	5
Visual Design & Oral Presentation	8
Total	60

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Project Title: Molecular Analysis of P450 Enzyme Kinetics

Objective:

Timeline:

Overview:

Project Flow:

What to Include in Your Poster

Title and Group Info (8 points)

Background/Objective (5 points)

Methods (5 points)

Results (39 points)

Figure 1. Protein Expression (From Project 3)

Figure 2. Determining the Km for enzyme and substrate

Figure 3. Determining the Ki of the inhibitor

Figure 4. Identifying the type of Inhibition

Discussion (3 points)

Presentation Expectations

1. Results - Experimental Results – 22 points

2. Results - Q&A and Understanding of Concepts – 17 points

3. Background & Discussion – 8 points

4. Methods (Flowchart Format) – 5 points

5. Presentation Quality & Visual Design – 8 points

Summary of Grading Criteria:

Background: Molecular Analysis of P450 Enzyme Reaction Rates

Project Title: Molecular Analysis of P450 Enzyme Kinetics

Objective:

Timeline:

Overview:

Project Flow:

What to Include in Your Poster

Title and Group Info (8 points)

Background/Objective (5 points)

Methods (5 points)

Results (39 points)

Figure 1. Protein Expression (From Project 3)

Figure 2. Determining the Km for enzyme and substrate

Figure 3. Determining the Ki of the inhibitor

Figure 4. Identifying the type of Inhibition

Discussion (3 points)

Presentation Expectations

Grading Rubric – Total: 60 points

1. Results - Experimental Results – 22 points

2. Results - Q&A and Understanding of Concepts – 17 points

3. Background & Discussion – 8 points

4. Methods (Flowchart Format) – 5 points

5. Presentation Quality & Visual Design – 8 points

Summary of Grading Criteria:

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