Name
AP Biology LAB 2
ENZYME CATALYSIS
OVERVIEW
In this lab you will:
1. observe the conversion of hydrogen peroxide (H2O2) to water and oxygen gas by the enzyme catalase,
and
2. calculate the rate of the enzyme-catalyzed reaction.
OBJECTIVES
Before doing this lab you should understand:
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the general functions and activities of enzymes;
the relationship between the structure and function of enzymes;
the concept of initial reaction rates of enzymes;
how the concept of free energy relates to enzyme activity;
that changes in temperature, pH, enzyme concentration, and substrate concentration can affect the
initial reaction rates of enzyme-catalyzed reactions; and
catalyst, catalysis, and catalase
After doing this lab you should be able to:
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measure the effects of changes in temperature, pH, enzyme concentration, and substrate concentration
on reaction rates of an enzyme-catalyzed reaction in a controlled experiment, and
explain how environmental factors affect the rate of enzyme-catalyzed reactions.
INTRODUCTION
In general, enzymes are important proteins produced by living cells; they act as catalysts in biochemical
reactions. A catalyst affects the rate of a chemical reaction. In some cases, enzymes increase the speed of
reactions that would otherwise occur slowly, and carry out complex chemical activities at relatively low
temperatures.
Enzymes are involved in nearly all metabolic processes. In an enzyme-catalyzed reaction, the substance to be
acted upon, the substrate (S), binds reversibly to the active site of the enzyme (E). 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 break down additional substrate
molecules. Each enzyme is specific for a particular reaction because its amino acid sequence is unique and
causes it to have a unique three-dimensional structure. 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. A description of several ways enzyme action may be affected follows:
1. Salt Concentration. If the salt concentration is close to zero, the charged amino acid side chains of
the enzyme molecules will attract each other. The enzyme will denature and form an inactive
precipitate. If, on the other hand, the salt concentration is very high, normal interaction of charged
groups will be blocked, new interactions will occur, and again the enzyme will precipitate. An
intermediate salt concentration, such as that of human blood (0.9%) or cytoplasm, is the optimum for
many enzymes.
2. pH. pH is a logarithmic scale that measures the acidity, or H+ concentration, in a solution. The scale
runs from 0 to 14 with 0 being the highest in acidity and 14 the lowest. When the pH is in the range of
0-7, a solution is said to be acidic; if the pH is around 7, the solution is neutral; and if the pH is in the
range of 7-14, the solution is basic. Amino acid side chains contain groups, such as –COOH and –NH2,
that readily gain or lose H+ ions, As the pH is lowered an enzyme will tend to gain H+ ions, and
eventually enough side chains will be affected so that the enzyme’s shape is disrupted. Likewise, as the
pH is raised, the enzyme will lose H+ ions and eventually lose its active shape. Many enzymes perform
optimally in the neutral pH range and are denatured at either an extremely high or low pH. Some
enzymes, such as pepsin, which acts in the human stomach where the pH is very low, have a low pH
optimum.
3. Temperature. Generally, chemical reactions speed up as the temperature is raised. As the
temperature increases, more of the reacting molecules have enough kinetic energy to undergo the
reaction. Since enzymes are catalysts for chemical reactions, enzyme reactions also tend to go faster
with increasing temperature. However, if the temperature of an enzyme-catalyzed reaction is raised
still further, a temperature optimum is reached; above this value the kinetic energy of the enzyme
and water molecules is so great that the conformation of the enzyme molecules is disrupted. The
positive effect of speeding up the reaction is now more than offset by the negative effect of changing the
conformation of more and more enzyme molecules. Many proteins are denatured by temperatures
around 40-50oC, but some are still active at 70-80oC, and a few even withstand boiling.
4. Activation and Inhibitors. Many molecules other than the substrate may interact with an enzyme.
If such a molecule increases the rate of the reaction it is an activator; and if it decreases the reaction
rate it is an inhibitor. These molecules can regulate how fast the enzyme acts. Any substance that
tends to unfold the enzyme, such as an organic solvent or detergent, will act as an inhibitor. Some
inhibitors act by reducing the –S-S bridges that stabilize the enzyme’s structure. Many inhibitors act by
reacting with side chains in or near the active site to change its shape or block it. Many well-known
poisons, such as potassium cyanide and curare, are enzyme inhibitors that interfere with the active site
of critical enzymes.
The enzyme used in this lab, catalase, has four polypeptide chains, each composed of more that 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 (H2O2) formed as a byproduct of metabolic processes.
Catalase might also take part is some of the many oxidation reactions that occur in all cells. Catalase speeds up
the breakdown of hydrogen peroxide into water (H2O) and oxygen gas (O2). The chemical equation for this
reaction is as follows:
2H2O2 (catalase)  2H2O + O2 (+ catalase)
In the absence of catalase, this reaction occurs spontaneously but very slowly. Catalase speeds up the reaction
considerably. In this experiment, a rate for this reaction will be determined.
Name
AP LAB 2: ENZYME CATALYSIS
STUDENT PROCEDURE
Label the enzyme, substrate, product A, and product B below specifically for this lab.
Enzyme: ___________________
Substrate __________________
Product A: ________________
Product B: _________________
Test 1:
CHANGE IN ENZYME CONCENTRATION…..to see what happens when you have a
substrate, and the amount of enzyme changes.
1. Obtain 4 test tubes and label with post its: 100%, 50%, 25%, and 0%.
2. Prepare 20 ml of enzyme for each of the above concentrations by using the following volumes in the
table below. You will use a graduated cylinder to measure the volumes.
Catalase enzyme stock (prepared by
Mrs. B)
*20 ml
10 ml
5 ml
0 ml
cold water
Percentage (%)
0 ml
10 ml
15 ml
20 ml
100
50
25
0
*you will save the undiluted enzyme (100 %) for Test 2
3.
4.
5.
6.
7.
8.
9.
10.
11.
Obtain 2 test tubes for the substrate (hydrogen peroxide).
Place 25 ml of 3% hydrogen peroxide solution into each test tube.
Use a hole punch to punch out 2 filter punches from a coffee filter.
Using forceps, immerse both filter punches in the 100% catalase enzyme solution for 5 seconds (use a
timer). Keep them separated so they don’t stick to each other.
After 5 seconds of immersion in the enzyme, remove the filters and drain by touching the edge to a
paper towel for 10 seconds. The filter punches now have 100% catalase on it.
Drop the punches (1 in each tube) that has 25 ml of 3% hydrogen peroxide making sure that it rests on
the bottom of the cup (use a stirring rod to push it down if necessary). Start the timer when it touches
the bottom.
In Table 1, record the time it takes for the disc to float to the top of the substrate (hydrogen peroxide)
solution.
Set aside the test tubes that have the used substrate (hydrogen peroxide)
Repeat steps 3-10 for the other filter disk to obtain a total of 2 trials for each change in enzyme
concentration. Do not reuse the hydrogen peroxide. (NOTE: Set aside the 100% catalase test tube to use
later in the lab). Now you are dipping disks into 50%, 25%, and 0% catalase solutions and
performing 2 trials for each and keeping the concentration of the substrate the same (3% hydrogen
peroxide).
12. Clean up by dumping everything EXCEPT the test tube that has 100% catalase in it (which you should
have already set aside).
13. Graph the data using averages.
Test 1 of the lab asks you to test the effect of enzyme concentration on enzyme activity. The enzyme
concentration will change, while the substrate concentration is kept constant. State your hypothesis:
TABLE 1:
Enzyme concentration
(%)
Disk 1
Floating Time (sec)
Disk 2
Floating Time (sec)
Average
Floating Time (sec)
100
50
25
0
Test 2:
CHANGE IN SUBSTRATE CONCENTRATION…..to see what happens when you have
the same enzyme concentration, and the amount of substrate changes.
Reminder: The substrate in the lab is ______________________________.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Obtain 8 test tubes and label with post its (2 of each): 0%, 3%, 10%, and 30% hydrogen peroxide.
Place 25 ml of the substrate (hydrogen peroxide) into the appropriately labeled test tubes.
Use a hole punch to punch out 2 filter punches from a coffee filter.
Using forceps, immerse both filter punches in the 100% catalase enzyme solution for 5 seconds (that
you saved from test 1). Keep them separated so they don’t stick to each other.
After 5 seconds of immersion in the enzyme, remove the filter and drain by touching the edges to a
paper towel for 10 seconds. The filter punches now have 25% catalase on it.
Drop the punches (1 in each test tube) into the 100mL of 0% hydrogen peroxide making sure that they
rest on the bottom of the tube. Use a stirring rod to push down if necessary. Start the timer when it
touches the bottom.
In Table 1, record the time it takes for each disc to float to the top of the substrate (hydrogen peroxide)
solution.
Repeat this procedure for the 3%, 10%, and 30% substrate (hydrogen peroxide) to see how the
change in hydrogen peroxide concentration affects the time to float by using the same concentration
(100%) of enzyme.
Record all data in Table 2 and be sure to calculate the average time for the two discs in each trial.
Graph the data using averages.
Test 2 of the lab asks you to test the effect of substrate concentration on enzyme activity. The substrate
concentration will change, while the enzyme concentration will remain constant. State your hypothesis:
TABLE 2:
Substrate
concentration (%)
Disk 1
Floating Time (sec)
Disk 2
Floating Time (sec)
Average
Floating Time (sec)
0
3
10
30
Test 3:
CHANGE IN TEMPERATURE CONCENTRATION…..to see what happens when you
have the same enzyme and substrate concentrations, but the temperature changes.
Reminder: The substrate in the lab is ______________________________.
Reminder: The enzyme in the lab is _______________________________.
1. Label 6 test tubes as follows with post its (2 of each): cold, room temp, and hot. Add 25 mL room temp
3% hydrogen peroxide to them. The hydrogen peroxide temperature is not being tested. The test is to
see if catalase holds up when exposed to altered temps.
2. At the side of the room, notice 2 water baths. One is set at a COLD temp and the other is HOT. Both
have 100% catalase solutions in them.
3. Use a hole punch to punch out 2 filter punches from a coffee filter.
4. Using forceps, immerse the 2 filter punches in the COLD TEMP catalase enzyme solution for 5 seconds.
Separate them with forceps if they stick to one another.
5. After 5 seconds of immersion in the enzyme, remove the filters and drain by touching the edges to a
paper towel for 10 seconds. The filter punches now have cold catalase on it.
6. Drop the punches (1 in each tube) into the test tube that has 3% hydrogen peroxide making sure that
they rest on the bottom of the cup. Use a stirring rod to push down if necessary. Start the timer when it
touches the bottom.
7. In Table 3, record the time it takes for each disc to float to the top of the substrate (hydrogen peroxide)
solution.
8. Repeat this procedure for the room temp and hot catalase to see how the change in temperature
affects the enzyme’s ability to break hydrogen peroxide down.
9. Record all data in Table 3 and be sure to calculate the average time for the two discs in each trial.
10. Graph the data using averages.
Test 3 of the lab asks you to test the effect of temperature on enzyme activity. The temperature the enzyme
is exposed to will change, while the enzyme concentration and substrate concentration remain constant. State
your hypothesis:
TABLE 3:
Temperature Catalase
was exposed to
Cold
Room temp
Hot
Disk 1
Floating Time (sec)
Disk 2
Floating Time (sec)
Average
Floating Time (sec)
QUESTIONS and ANALYSIS:
1. How is “enzyme activity” for this lab experiment being measured? Explain.
2. What are the dependent and independent variables in TEST 1?
3. How does enzyme activity vary with enzyme concentration?
4. In Test 1, what is kept constant throughout this test?
5. In Test 1, what variable is being tested?
6. What are the dependent and independent variables in TEST 2?
7. What do you think would happen if you increased the substrate concentration to 50% hydrogen
peroxide? Would you see a change in your graph?
8. In Test 2, what is kept constant throughout the experiment?
9. In Test 2, what variable is being tested?
10. Does changing the substrate concentration exhibit the same effect as changing the enzyme
concentration?
11. Many enzymes that participate in reactions in the human body work best under normal body
temperatures. How is this enzyme derived from potato extract different from an enzyme found in the
human body?
12. Assume the following results were obtained. Catalase was exposed to varying concentations of pH.
pH Level
disc 1
Time (s)
disc 2
Average
pH 4
pH 6
pH 7
pH 8
pH 9
13. Using the graph paper provided, graph the data shown in #12.
14. What conclusions can you draw on the optimum pH level for the enzyme peroxidase?
15. Would you assume that most all enzymes used in the human body prefer to be in a neutral
environment? If not, give an example of where you may find enzymes in the human body that prefer
pH’s outside the neutral range.