Enzyme Kinetics: Investigating Amylase Activity on Starch

Experiment 5: Enzyme Kinetics - Investigating the Activity of Amylase on Starch

Introduction

  • Enzyme kinetics is a crucial branch of biochemistry that studies:

    • The speed of enzyme-catalyzed reactions
    • Factors influencing reaction rates

  • Enzymes function as biological catalysts:

    • Increase the rate of biochemical reactions
    • Lower the activation energy without being consumed

  • Models of enzyme-substrate interactions:

    • "Lock and Key" model: Enzyme provides a specific structure for substrate binding.
    • "Induced Fit" model: Enzyme changes shape to accommodate the substrate, enhancing the reaction.

  • Michaelis-Menten equation:

    • Describes the relationship between reaction velocity (speed) and substrate concentration.
    • Key parameters:
    • Maximum reaction rate (Vmax)
    • Michaelis constant (Km)

  • Importance of enzyme kinetics:

    • Understanding metabolic regulation.
    • Drug interaction with enzymes.
    • Insights into metabolic disorders.
    • Applications in biotechnology and medicine.

Learning Objectives

  • Understand fundamental principles of enzyme kinetics and its biochemical importance.
  • Describe the role of enzymes as catalysts, and their impact on reaction rates along with affecting factors.
  • Explain enzyme-substrate interactions with models like “lock and key” and “induced fit.”
  • Apply the Michaelis-Menten equation for analyzing reaction velocity versus substrate concentration.

Materials Needed

  • Lab Apparatus/Equipment:

    • 16 Test tubes (per group)
    • Test tube rack (1 per group)
    • Spotting plate (1 per group)
    • Two 10ml graduated cylinders (2 per group)
    • Dropper (2 per group)

  • Chemicals:

    • 1% Starch solution
    • Lugol's iodine
    • Buffer solutions at different pH (4, 7, 10)
    • Examples: Acetic acid and sodium acetate, Phosphate buffer, Sodium bicarbonate or borate buffer

Experimental Procedures

A. Acquisition of Salivary Amylase
  1. Collect saliva from a volunteer (mandatory fasting for 30 mins).
  2. Gather at least 20mL into a test tube.
  3. Use a dropper to extract samples for further procedures.
B. Enzyme & Substrate Concentration
  1. Prepare three test tubes with varying amounts of starch:
    • Test tube 1: 10 mL 1% starch
    • Test tube 2: 5 mL water + 5 mL 1% starch
    • Test tube 3: 9 mL water + 1 mL 1% starch
  2. Label and add 1 drop of Lugol's iodine in each row of the spotting plate.
  3. Add 1 mL of saliva to each test tube.
  4. At one-minute intervals for 5 minutes, take a drop from each test tube and test with Lugol's iodine to observe changes.
C. Changes in pH
  1. In three test tubes, mix 2.5 mL buffer with 2.5 mL of 1% starch solution; label accordingly by pH.
  2. Add 2-3 drops of Lugol's iodine.
  3. Add 1 mL of saliva, mix by shaking (do not heat).
D. Temperature
  1. Prepare four test tubes labeled as follows:
    • A: 0°C
    • B: 25°C
    • C: 37°C
    • D: 60°C
  2. Add 2.5 mL of 1% starch and 2.5 mL saliva to each test tube.
  3. Place test tubes in corresponding environments: freezer, room temp, and oven.
  4. Monitor for 10 minutes and check every 5 minutes for reaction status.
  5. Record the time for partial/complete hydrolysis indicated by the absence of blue-black color.

Data and Results

B. Enzyme & Substrate Concentration Data
  • Analyze changes per interval for each sample solution for observable results and interpretations.
C. Changes in pH Data
  • Document color reaction with iodine for various buffer pH levels (4, 7, and 10).
D. Temperature Data
  • Note the color reaction at different temperatures (0°C, 25°C, 37°C, 60°C).
  • Record total time for complete hydrolysis.

Questions for Research

  1. Explore the effect of varying substrate concentrations on the reaction rate of ptyalin-catalyzed starch hydrolysis.
  2. Investigate how temperature fluctuations alter the stability and efficiency of ptyalin and the molecular mechanisms causing enzyme denaturation at higher temperatures.
  3. Analyze the variation in enzyme-substrate affinity and reaction kinetics with competitive vs non-competitive inhibitors and their implications for enzyme regulation in metabolic pathways.

References

  • Fastrez, J., & Fersht, A. R. (1973). Enzyme kinetics based on free-energy profiles. Biochemistry, 12(11), 2025–2034. https://doi.org/10.1021/bi00735a001
  • Cornish-Bowden, A. (2004). Enzyme kinetics. Portland Press.
  • Bisswanger, H. (2015). Enzyme kinetics. Wiley-VCH.