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Lab 13: Enzyme Catalysis
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AP Biology
Name________________________
(from Ellis – AP biology)
Lab 13: Enzyme Catalysis1
Overview
In this lab you will:
1.
2.
observe the conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the enzyme
catalase, and
measure the amount of O2 produced and calculate the rate of the enzyme-catalyzed reaction.
Objectives
Before doing this lab you should understand:
1. the general functions and activities of enzymes
2. the relationship between structure and function of enzymes
3. the concept of initial reaction rates of enzymes
4. how the concept of free energy relates to enzyme activity
5. the meaning of the words catalyst, catalysis, and catalase
After doing this lab you should:
1. understand the relationship between enzyme structure and function
2. be able to make some generalizations about enzymes by studying just one enzyme in particular
3. be able to determine what factors can change the rate of an enzymatic reaction
4. be able to determine which factors that affect enzyme activity could be biologically important
Introduction
In general, enzymes are proteins produced by living cells; they act as catalysts in biochemical
reactions. A catalyst increases the rate of a chemical reaction. One consequence of enzyme activity is that
cells can carry out complex chemical activities at high rates at relatively low temperatures.
In an enzyme-catalyzed reaction, the substance to be acted upon, the substrate (S), binds reversibly
to the active site of the enzyme (E), forming a temporary enzyme-substrate complex. One result of this
temporary union is a reduction in the energy required to activate the reaction of the substrate molecule so
that the products (P) of the reaction are formed. In summary:
E + S  ES  E + P
Note that the enzyme is not changed in the reaction and can be recycled to act upon additional substrate
molecules.
Enzymes have specific shapes and structures that determine their functions. The enzyme’s active site
is very selective, allowing only certain substances to bind. If the shape of an enzyme is changed in any
way, or the protein denatured, then the binding site also changes, thus disrupting enzymatic functions. The
active site is the portion of the enzyme that interacts with the substrate, so that any substance that blocks or
changes the shape of the active site affects the activity of the enzyme.
Enzymes are fundamental to the survival of any living system and are organized into a number of
groups depending on their specific activities. Two common groups are catabolic enzymes (“cata” or
“kata” from the Greek “to break down”)—for instance, amylase breaks complex starches into simple
sugars—and the anabolic enzymes (“a-” or “an-” from the Greek “to build up”). (You may know this
second word already from stories about athletes who have been caught using anabolic steroids to build
muscle.)
1
Adapted from the College Board AP Biology Student Lab Manual, 2001 edition and 2012 edition, and a
lab produced by Kim Foglia.
Lab 13: Enzyme Catalysis
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Catalytic enzymes called proteases break down proteins and are found in many organisms; one
example is bromelain, which comes from pineapples and can break down gelatin. Bromelain often is an
ingredient in commercial meat marinades. Papain is an enzyme that comes from papaya and is used in
some teeth whiteners to break down the bacterial film on teeth. People who are lactose intolerant cannot
digest milk sugar (lactose); however, they can take supplements containing lactase, the enzyme they are
missing. All of these enzymes hydrolyze large, complex molecules into their simpler components;
bromelain and papain break proteins down to amino acids, while lactase breaks lactose down to simpler
sugars.
Anabolic enzymes are equally vital to all living systems. One example is ATP synthase, the
enzyme that stores cellular energy in ATP by combining ADP and phosphate. Another example is rubisco,
an enzyme involved in the anabolic reactions of building sugar molecules in the Calvin cycle of
photosynthesis.
The enzyme used in this lab, catalase, has four polypeptide chains, each composed of more than
500 amino acids. This enzyme is ubiquitous in aerobic organisms. One function of catalase within cells is
to prevent the accumulation of toxic levels of hydrogen peroxide, which is formed as a by-product of
metabolic processes. Catalase may also take part in some of the many oxidation reactions that occur in all
cells.
The primary reaction catalyzed by catalase is the decomposition of H2O2 to form water
and oxygen:
2 H2O2  2H2O + O2 (gas)
In the absence of catalase, this reaction occurs spontaneously but very slowly. Catalase speeds up
the reaction considerably. In this lab, you will determine a rate for this reaction.
Much can be learned about enzymes by studying the kinetics (particularly the changes in rate) of
enzyme-catalyzed reactions. For example, it is possible to measure the amount of product formed, or the
amount of substrate used, from the moment the reactants are brought together until the reaction has
stopped.
Let’s look at a hypothetical example. In this case, an enzyme and its substrate are mixed in a
reaction vessel. If the amount of product formed is measured at regular intervals and the quantity is plotted
on a graph, a curve like the one in Figure 1 is obtained.
Study the solid line on the graph of this reaction. At time 0 there is no product. After 30 seconds,
5 micromoles (moles) have been formed; after 1 minute, 10 moles; after 2 minutes, 20 moles. The rate
of this reaction could be given as 10 moles of product formed per minute for this initial period. Note,
however, that by the third and fourth minutes, only about 5 additional moles of product have been formed.
During the first three minutes, the rate is constant. From the third minute through the eighth minute, the
rate is changing; it is slowing down. For each successive minute after the first three minutes, the amount of
product formed in that interval is less than in the preceding minute. From the seventh minute onward, the
reaction rate is very slow.
Lab 13: Enzyme Catalysis
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In the comparison of the kinetics of one reaction with another, a common reference point is
needed. For example, suppose you wanted to compare the effectiveness of catalase obtained from potato
with that of catalase obtained from liver. It is best to compare the reactions when the rates are constant. In
the first few minutes of an enzymatic reaction such as this, the number of substrate molecules is usually so
large compared with the number of enzyme molecules that changing the substrate concentration does not
(for a short period at least) affect the number of successful collisions between substrate and enzyme.
During this early period, the enzyme is action on substrate molecules at a nearly constant rate. The slope of
the graph line during this early period is called the initial rate of the reaction. The initial rate of any
enzyme-catalyzed reaction is determined by the characteristics of the enzyme molecule. It is always the
same for any enzyme and its substrate at a given temperature and pH. This also assumes that the substrate
is present in excess.
The rate of the reaction is the slope of the linear portion of the curve. To determine a rate, pick
any two points on the straight-line portion of the curve. Divide the difference in the amount of product
formed between these two points by the difference in time between them. The result will be the rate of the
reaction which, if properly calculated, can be expressed as moles of product/sec. The rate, then, is:
(moles2 – moles1) ÷ (t2 – t1) or, from the graph, y ÷ x
In the illustration if Figure 2.1, the rate between two and three minutes is calculated:
(30 mol – 20 mol) ÷ (180 sec. – 120 sec.) = 10 mol ÷ 60 sec. = 0.17 mol/sec
The rate of a chemical reaction may be studied in a number of ways, including the following:
1. measuring the rate of disappearance of substrate (in the case of catalase, H 2O2)
2. measuring the rate of appearance of product (in the case of catalase, O2 gas is the easiest to
measure, and this is what we will do)
3. measuring the heat released (or absorbed) during the reaction (this is difficult to do)
Key Vocabulary
Baseline is a universal term used in studying most chemical reactions. In this investigation, the
term is used to describe a standard for a reaction. Thus, when manipulating components of a reaction, you
have a reference reaction for comparison to help understand what happened in the experimental reaction.
The baseline may vary with different scenarios pertinent to the design of the experiment, such as altering
the environment in which the reaction occurs. In this scenario, different conditions may be compared, and
the effects of changing an environmental variable (e.g., pH) can be determined.
Part A1 – Introduction to Catalase Activity
Before coming to lab
1.
2.
Read through the procedure thoroughly.
Prepare your lab notebook by writing out the procedure in your own words or pictures (in a format
that’s easy for you to follow) and leaving spaces for your observations and answers to questions.
Procedure
1.
To observe the reaction to be studied, transfer 10 mL of 1.5% (0.44 M) H2O2 into a small glass
beaker or clear plastic cup and add 1 mL of the freshly made catalase solution. Record
observations. The bubbles coming from the reaction mixture are O2, which results from the
breakdown of H2O2 by catalase. Answer the following questions in your lab notebook. Clearly
indicate what questions you are answering (either copy the question or give a full sentence answer
that indicates what the question asked) and what you did before answering the questions.
a. What is the enzyme in this reaction?
b. What is the substrate in this reaction?
c. What is the product in this reaction?
Lab 13: Enzyme Catalysis
2.
3.
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d. How could you show that the gas in the bubbles is O 2? Describe direct evidence that could
be collected.
To demonstrate the effect of boiling on enzymatic activity, transfer 5 mL of the catalase solution
to a test tube and place it in a boiling water bath for 5 minutes. Then allow the catalase to cool to
room temperature or below (using ice). Transfer 10 mL of 1.5% H2O2 into a small beaker or cup
and add 1 mL of the cooled, boiled catalase solution. Answer the following in your lab notebook:
How does the reaction compare to the one using the unboiled catalase? Explain the reason for this
difference.
To demonstrate the presence of catalase in living tissue, cut around 1 cm3 of potato, chop it up,
and transfer to a beaker or cup containing 10 mL of 1.5% H2O2. Record your observations, and
indicate what you think would happen if the potato were boiled and then cooled before being
added to the H2O2. Be sure to label your response so that anyone reading your lab notebook can
understand it.
Part A2: Test of Catalase activity - General Lab Setup and Baseline Data Collection:
Before coming to lab
1. Read through the procedure thoroughly.
2. Prepare your lab notebook by writing out the procedure in your own words or pictures (in a format
that’s easy for you to follow).
3. Set up a data table that you can use. Be sure to give it a title and include a space for the reading at
time = 0 min.
Materials
50 mL beaker with fresh catalase (yeast) solution
reaction chamber
ring stand
2 ring stand clamps
10 mL graduated cylinder
gas collection tube (eudiometer)
3% hydrogen peroxide (H2O2)
pan (water bath)
pipette
thermometer
hot plate
Procedure
1.
2.
3.
4.
5.
6.
7.
8.
At your lab bench you will find a round vial with a rubber stopper that fits in the top. This will be referred to as the
reaction chamber. You will also find a eudiometer (a gas collection tube), a ring stand and clamp, and a plastic pan
which will be used as a water bath. All of the equipment needs to be assembled into the experimental apparatus as
described and illustrated below.
Fill the pan ¾ full using tap water. Allow the water to come to room temperature.
Fill the eudiometer completely with water. Put your thumb over the open end of the eudiometer and turn it upside
down, keeping the open end underwater, so that they eudiometer remains filled with water. Suspend the eudiometer
upside down and with its open end in the water using the clamp and the ring stand. Adjust the height of the clamp on
the ring stand so that the open end of the eudiometer is about 3 cm above the bottom of the pan. The picture on the
next page will help you set up this apparatus, although the picture shows a graduated cylinder instead of a eudiometer.
Place a thermometer in the pan so that you can monitor the temperature of the water.
Put 10 mL of 3% H2O2 solution in the reaction chamber.
Add 1.0 mL of the catalase (yeast) solution and IMMEDIATELY stopper the reaction chamber tightly, submerge it in
the water bath, and adjust the plastic tubing so that any bubbles formed in the reaction chamber will rise into the
eudiometer. Note that you may need to hold the reaction chamber in place throughout the reaction. A free ring stand
clamp may be used for this purpose. Throughout, try to keep the reaction chamber flat on the bottom of the water bath
so that the liquids inside the reaction chamber do not enter the tubing.
As soon as the reaction chamber is in place, start a timer.
Measure the gas levels in the eudiometer at 30-second intervals for 5 minutes.
stand so the open end of the graduated cylinder is about 3cm above the bottom of the pan.
See diagram below.
5. Place a thermometer in the pan and record the temperature
of the water, during Part A of the lab.
_____________°C
6.Lab
When
sections of
the lab are complete, share the data with the class from your group’s
13:all Enzyme
Catalysis
section. Each person must plot the data for all parts of the lab on his/her own graph paper.
p. 5
Data Analysis
Plot the data for your time course on a graph. Be sure to give it a good title and indicate that it is from the general lab
4 of 11 Review proper graphing methods if necessary. If your reaction chamber produced
procedure. In fact, it is your baseline!
2007-2008
gas beyond the volume you were able to measure, your curve should just end at the first data point at the maximum volume
you could measure. Do not continue the curve beyond where you have data.
Part B: The Effect of pH on Catalase Activity
Before coming to lab
1.
2.
3.
Copy the procedure below into your lab notebook using your own words or pictures. Be sure that you have indicated
that your procedure is for Part B.
Make a special note for yourself to use 6% H2O2 when you make your solutions, not 3%.
Set up a data table for Part B. Be sure to give it a title and to include a line for time 0.
Procedure
Repeat the experiment from Part B (5 min run with 1 mL of catalase solution) three different times, each time using 10 mL
of a 3% H2O2 solution at a different pH.
a. pH 4: Make a 3% solution of H2O2 at pH 4 by adding 5 mL of 6% H2O2 to 5 mL of pH 4 buffer.
b. pH 7: Make a 3% solution of H2O2 at pH 7 by adding 5 mL of 6% H2O2 to 5 mL of pH 7 buffer.
c. pH 10: Make a 3% solution of H2O2 at pH 10 by adding 5 mL of 6% H2O2 to 5 mL of pH 10 buffer.
d. pH 12: Make a 3% solution of H2O2 at pH 12 by adding 5 mL of 6% H2O2 to 5 mL of pH 12 buffer,
Record all of your data in your data table.
Data Analysis
Plot the data from Part B on a new graph. Clearly label the graph as Part B, and don’t forget to label the axes and plotted
lines, and to title the graph.
Inquiry: Designing and Conducting Your Own Investigation
You now have the basic information and tools needed to explore enzymes in more depth on your own. In this part
of the lab, you will do just that. You will have the chance to develop and test your own hypotheses about enzyme activity.
To help you get started, read the following questions, and write your answers in a well-labeled spot in your lab notebook.
 In Part A, was the limiting factor in your baseline reaction the amount of enzyme or the amount of substrate? How
could you use the procedure you learned to answer this question?
Lab 13: Enzyme Catalysis
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What are three or four factors that vary in the environments in which organisms live? Which of these factors do
you think could affect enzyme activity? How could you modify the basic assay to test your hypotheses?
You should now design and conduct an experiment to investigate an answer(s) to one of the questions above or another
question that might have been raised for you as you completed Parts A and B. Remember, the primary objective is to
explore how biotic and abiotic factors influence the rate of enzymatic reactions.
You should do the following:
 Write your hypothesis in your lab notebook and clearly identify it as a hypothesis.
 Write out the procedure you followed in enough detail that another person could replicate your procedure. It’s OK
to just write out that you modified the general procedure and be specific about the modifications without repeating
the details in the general procedure that you left unchanged.
 Record your data in a proper data table.
 Graph your data appropriately.
 Indicate whether the data support or do not support your hypothesis.
 Attempt to explain the effect of the environmental factor you tested on the rate of your enzymatic reaction. Why
does the curve in your graph have the shape it does?
Analysis Questions
Answer the following in your own words in your lab notebook. Everyone must answer every question, even though you
did not do all parts of the lab.
1.
2.
3.
4.
5.
If you wanted to determine the rate of enzyme activity from any part of this lab, how would you do it?
The data from Part B show the overall effect of pH. Calculate the initial rate for your reactions in Part B. This should
give you four values for initial rate. Then graph initial rate of reaction vs. pH. Use proper graph format.
What can you conclude based on the graph you just made?
What would a similar graph (initial rate vs. your independent variable) from your self-designed experiment look like,
and why?
After an enzyme is mixed with its substrate, the amount of product formed is determined at 10-second intervals for 1
minute. Data from the experiment are shown below:
time (sec)
product formed (mg)
a.
b.
c.
d.
0
0.00
10
0.25
20
0.50
30
0.70
40
0.80
50
0.85
60
0.85
What is the initial rate of the enzymatic reaction? Show your calculations.
What is the rate after 50 seconds? Show your calculations.
Why is the initial rate different from the rate after 50 seconds?
Think about how the rate changes over time. Make a simple sketch of what you would see if you graphed rate
of reaction over time. Your graph does not need a title or numbers on the axes, but it should have labels on
the axes and the curve should be the correct shape.
PRELAB HW:
Prelab – start on new page – title & date
Part A1 – test of catalase activity
p. 3: prelab – (#1-2) allow at least 1 page
Part A2- baseline data collection
p. 4 : prelab (#1-3)
start on new page, leave one full blank page after data table for graph
Part B – effect of pH
p. 5: prelab (#1-3)
start on new page