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Lab manual : Protein Biomanufacturing and Biochemistry (Biot274 @IVC)
10: Activity 3-1 - Michaelis-Mentent Analysis of Substrate Concentration on Reaction Rate

10: Activity 3-1 - Michaelis-Mentent Analysis of Substrate Concentration on Reaction Rate

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Jul 17, 2025
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- 9: Activity 3-0 - Project Instructions - Investigating Enzyme Kinetics
- 11: Activity 3-2 - Determining the IC₅₀ of Inhibitor

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Learning Objectives

Define key terms related to enzyme kinetics, including substrate, velocity, V_max, and K_M.
Explain the relationship between substrate concentration and enzymatic reaction rate.
Describe the purpose of the Michaelis-Menten equation.
Apply Beer’s Law to calculate enzyme concentration from spectrophotometric data.
Recognize why optimizing enzyme and substrate concentrations is essential in kinetic studies.

Definition: Term

Enzyme: A biological catalyst that speeds up chemical reactions without being consumed.
Substrate: The specific molecule upon which an enzyme acts.
Product: The molecule(s) formed from substrate transformation.
Reaction Rate (Velocity): The speed at which the product is formed, typically measured as Δ[product]/Δtime.
V_max: The maximum rate of reaction when the enzyme is fully saturated with substrate.
K_M: The substrate concentration at which the reaction rate is half of V_max.
Michaelis-Menten Equation: Describes how reaction velocity varies with substrate concentration.
Beer’s Law: A mathematical relationship used to determine concentration from absorbance: A = ε × c × l.
Extinction Coefficient (ε): A constant that indicates how strongly a substance absorbs light at a specific wavelength.
Cytochrome P450: A family of heme-containing enzymes involved in metabolizing fatty acids, drugs, and toxins.

Pre-Lab Questions

What does it mean for an enzyme to be saturated with substrate?
Why is K_M considered a measure of substrate affinity?
What are the units for V_max and K_M?
Explain how absorbance at 418 nm is used to determine P450 concentration.
What could happen if you use an enzyme concentration that is too high or too low?

In-Lab Focus

Use Beer’s Law to calculate enzyme concentration.
Vary both enzyme and substrate concentrations to observe their effects on reaction rate.
Generate a Michaelis-Menten plot and estimate kinetic parameters (V_max and K_M).

Effect of Substrate Concentration on Reaction Rate

Background

In this lab series, you will analyze the enzymatic activity of a cytochrome P450 enzyme, a heme-containing protein involved in the metabolism of fatty acids, drugs, and other compounds. One of the most fundamental properties of any enzyme is its kinetics—how fast it catalyzes a reaction under different conditions. Understanding the enzyme’s kinetics helps us characterize its efficiency, identify optimal reaction conditions, and compare its behavior under different physiological or experimental settings.

In this specific activity, you will investigate how substrate concentration affects the rate of the enzymatic reaction. This relationship is described by the Michaelis-Menten equation, which helps us determine two key kinetic parameters:

V_max: The maximum velocity the enzyme can achieve.
K_M: The substrate concentration at which the reaction rate is half of V_max.

Figuring out the best substrate concentration is critical. If the concentration is too low, the enzyme won’t function at its full potential. If it’s too high, you may saturate the enzyme, leading to wasted substrate and skewed data. By conducting this experiment and constructing a Michaelis-Menten plot, you will identify the optimal range of substrate concentrations and better understand how this enzyme behaves under physiological conditions.

Before testing substrate concentration effects, you’ll also learn how to calculate and optimize the amount of enzyme to use per reaction. You’ll do this by applying Beer’s Law to spectrophotometric data at 418 nm—an absorbance peak corresponding to the heme group in P450. This enables you to quantify the enzyme concentration accurately because each enzyme molecule contains one heme group.

Materials

All materials used since the beginning of lab
100 mM potassium phosphate buffer, pH 7.4 (from 1 M stock) OR 0.5X PBS
10 mM lauric acid in 50 mM potassium carbonate (Carolina #871840)
10 mM NADPH (Fisher #481973100MG)
Proteins (from previous experiments)
UV-Visible Spectrophotometer (Thermo Scientific #840-300000)
UVette cuvettes (Eppendorf #952010051)
Micropipettes and sterile tips
Parafilm

Procedures: Determine optimal Enzyme concentration for Kinetics Experiments

Thaw your protein samples on ice. Avoid repeated freeze-thaw cycles by preparing multiple aliquots.
Use Beer’s Law to calculate the concentration of your enzyme (Follow Activity 1.3):
- [Enzyme in nM] = [(Absorbance at 418 nm) ÷ (91,000M⁻¹cm⁻¹)] ÷ 10⁻⁹
Based on this concentration, calculate the volume (in µL) needed to add the following amounts of enzyme to your cuvettes:
- 0 nanomoles/cuvette → ______ µL
- 25 nanomoles/cuvette → ______ µL
- 50 nanomoles/cuvette → ______ µL
- 75 nanomoles/cuvette → ______ µL
- 100 nanomoles/cuvette → ______ µL
- 150 nanomoles/cuvette → ______ µL
Set up six (6) cuvettes and follow the protocol from Activities 1-4. Make sure to adjust the volume of Enzymes and buffer for each cuvette tested.
Determine the optimal enzyme concentration—Plot O.D. (absorbance) vs. time for each condition, and then look for the steepest AND linear increase over the 2-minute interval.

Procedures: Determine Km of substrates for Kinetics Experiments

Concerns: If you are low on Enzymes volumes, skip this experiment. Only go back here if you have any remaining enzyme after you determine your inhibitor type. Alternatively, you can decrease your enzyme volume by half or by a 3rd.

Calculate the volume (in µL) needed to add the following amounts of 10mM Lauric Acid (substrate) to your cuvettes:
- 0.05 mM (Optional) → ______ µL
- 0.1 mM → ______ µL
- 0.2 mM → ______ µL
- 0.4 mM → ______ µL
- 0.8 mM → ______ µL
- 1.0 mM → ______ µL
- 2.0 mM → ______ µL
Set up six (6) cuvettes (or more if you want to do the additional optional steps).
- Follow the protocol from Activities 1-4.
  - You will use the Optimal enzyme concentration volume you found here.
  - You will be adjusting the volume of Lauric Acid (substrate) and buffer for each cuvette tested.
  - Note: If you are low in enzymes, cut the enzyme volume in half.
Determine the Velocity—Plot Velocity vs. substrate concentration to create Michaelis-Menten plots
- Estimate Vmax and Km. The Km concentration will be what you will use for the next experiment.

Discussion Questions

Which enzyme concentration gave you the most linear rate of product formation? Why is that important?
How did the velocity of your enzyme change with increasing substrate concentration?
Was there a point where increasing substrate concentration had little or no effect on velocity? What does this indicate?
How well did your experimental data fit the Michaelis-Menten model?
What might cause deviations from the ideal curve (e.g., substrate inhibition, experimental error)?
How would you design a follow-up experiment to test enzyme inhibition?
Write a short paragraph (4–6 sentences) reflecting on the importance of choosing the correct substrate concentration when studying enzyme kinetics. What insights did you gain from plotting your own Michaelis-Menten curve?
Using your velocity and substrate concentration data, linearize your Michaelis-Menten results using a Lineweaver-Burk. Compare the V_maxand K_M values obtained from linear and nonlinear methods. Predict how a competitive or noncompetitive inhibitor would affect the shape of your curve. How could you design an experiment to test this?