Lab 2: Enzyme Catalysis
Introduction:
  • Enzymes catalyze a reaction by lowering the activation energy required for the reaction to take place.
  • The substrate is the molecule that an enzyme acts on
  • Enzymes are specific
    • In this lab, the enzyme is catalase, the substrate is hydrogen peroxide, and the products are water and oxygen.
    • the way the amino acids are in order at the active site makes it specific for one specific enzyme
  • Structure of an enzyme
    • globular protein
    • the way the enzyme is folded leaves a binding side for other molecules, known as the active site
enzyme_structure.gif
    • When an enzyme binds to the correct substrate a slight conformational change occurs. This is called an induced fit. This helps in the advancement of catalysis as the enzyme converts substrate to product.
  • Factors that effect enzyme catalysis
    • pH and temperature are two factors that can cause the denaturation of an enzyme. Therefore, it is no longer functional.
    • Enzyme catalysis speeds up as the temperature increases. Since every enzyme has an optimum temperature, surpassing this temperature can result in denaturization.
      • Boiling temperatures will denature most enzymes!
    • pH is an important factor because certain enzymes work better under a certain pH- an optimal pH
      • The enzyme pepsin that breaks down proteins in the stomach works best at a very acidic pH, whereas the enzyme lipase which breaks down fats in the small intestine works at a very basic pH

The Experiment

  • To observe the rate at which catalase catalyzes converts substrate to product.

  • Catalase reacted with hydrogen peroxide for various amounts of time.

  • In order to know how much hydrogen peroxide is remaining after the reaction a titration can be performed with the use of KMnO4.

    • You add this until a color change occursenzexper.gif

  • titration performance

    • slowly adding KMnO4 to the flasks causes the color change. When all the peroxide has reacted with KMnO4, the additional LMnO4 will be light brown or pinkish. This means it has reached its end point. The amount of KMnO4 should be recorded.

Important Hints:

1.

Be sure to titrate only 5 ml of the sample at a time. This way, if you exceed the endpoint or have an error in titration, you will have sufficientsample to repeat the titration.

2.

Since you will be comparing amounts of H2O2 remaining in the sample after different reaction times, be sure all your samples are the same size (5 ml).

3.

Place the solution to be titrated over a piece of white background paper so you can see the color changes easily.

4.

Swirl (do not shake) the flask after every few drops to mix well.

5.

For each assay, be sure to stop the titration at the same color.measure.gif

Analysis:

  • The rate of reaction is calculated by taking the change in y over the change in x. There are many different forms of the change over time graphs and they are all read differently. In this graph the appearance of product is what is being measured, but in our lab the disappearance of substrate was being measured.

timegr.gif

http://www.phschool.com/science/biology_place/labbench/lab2/analysis.html