AP Biology Lab 2 Enzyme Catalysis
Introduction
Enzymes catalyze reactions by lowering the activation energy necessary for a reaction to occur.
In this laboratory, you will study some of the basic principles of
molecuar movement in solution and perform a series of activities
to investigate these processes.
Key Concepts
Enzymes catalyze reactions by lowering the activation
energy necessary for a reaction to occur. The molecule that an
enzyme acts on is called the substrate. In an enzyme-mediated
reaction, substrate molecules are changed, and product is
formed. The enzyme molecule is unchanged after the reaction,
and it can continue to catalyze the same type of reaction over
and over.
Each enzyme is specific for the reaction it will catalyze. In this laboratory,
Enzyme = catalase
Substrate = hydrogen peroxide (H2O2)
Products = water and oxygen
H2O2
H2O + O2
If a small amount of catalase is added to hydrogen peroxide, you
will be able to observe bubbles of oxygen forming.
Over the next several screens we will take a closer look at what
happens during an enzymatic reaction. We begin with a model of
enzyme structure.
Enzyme Structure
Enzymes are globular proteins. Their folded conformation creates an area known as the active site. The nature and arrangement of
amino acids in the active site make it specific for only one type of substrate.
Binding Specificity
Even when different substrate molecules are present, only those that have the
specific shape complementary to the active site are able to bind with the enzyme's
active site.
Induced Fit
When an enzyme binds to the
appropriate substrate, subtle
changes in the active site
occur. This alteration of the
active site is known as an induced fit. Induced fit enhances catalysis, as the
enzyme converts substrate to product.
Release of the products restores the enzyme to its original form. The enzyme
can repeat this reaction over and over, as long as substrate molecules are
present.
Design of the Experiment
In this experiment, you will investigate the rate at which the enzyme catalase converts substrate to product. You will allow catalase
to react with hydrogen peroxide for varying amounts of time and then stop the reactions by adding H 2SO4.
To determine the amount of hydrogen peroxide that remains after the reaction, you will do a titration with KMnO 4. In such a
titration, you slowly add a chemical (KMnO4) that will cause a color change until a target color is achieved.
The following illustration shows you an overview of the procedure.
Doing the Titration
To determine how much
hydrogen peroxide (substrate)
has been broken down by
catalase at varying times, you
measure the amount of peroxide
remaining in each flask.
You slowly add KMnO4, which is
purple, to the flask. The
peroxide in the flask causes the
KMnO4 to lose color when the
solution is mixed thoroughly.
When all the peroxide has
reacted with KMnO4, any
additional KMnO4 will remain
light brown or pinkish even after
you swirl the mixture. This is the
endpoint. Record the amount of
KMnO4 you have used. (The
more KMnO4 you use, the more
peroxide is in the flask.)
Lab
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 sufficient sample 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.
Reading a Burette
Assume that the burette is filled to the point indicated in the figure at
the left. You would record the initial point as 3.30 ml; the ending
point would be 3.90 ml. Therefore, the titration would have required
0.60 ml. Remember that you should read the number that is at the
bottom of the meniscus.
Analysis of Results
Enzyme Action Over Time
We can calculate the rate of a reaction by measuring, over time, either the
disappearance of substrate (as in our catalase example) or the appearance of
product (as in the above graph). For example, on the graph above, what is the rate,
in moles/second, over the interval from 0 to 10 seconds?
so for this example, the rate would be