ENZYME LAB INVESTIGATION
Table of Contents
BACKGROUND ………………………………………………………………………………………. 2
SAFETY PRECAUTIONS …………………………………………………………………………… 8
PART 1: STRUCTURED INQUIRY — ESTABLISHING A BASELINE……………….. 9
PART 2: GUIDED INQUIRY — VARIABLES THAT AFFECT THE
RATE OF ENZYME REACTION …………………………………………………………………. 12
PART 3: OPEN INQUIRY - DESIGN AN EXPERIMENT …………………………………. 15
MATERIAL SAFETY DATA SHEETS …………………………………………………………… 17
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1
AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND
OBJECTIVES

Design a plan for collecting
data to show that all
biological systems are
affected by complex biotic
and abiotic interactions.

Use models to predict and
justify that changes in the
subcomponents of a
biological polymer affect the
functionality of the molecule.

Analyze data to identify how
molecular interactions affect
structure and function.
Enzymes, Substrates, and Products
Chemical reactions underlie metabolism. Organisms have evolved catalytic
proteins, called enzymes that can make the reactions more efficient by
lowering the activation energy of a chemical reaction. Catalytic efficiency of
enzymes is dependent upon the precise shape (conformation) of the active site
in the protein that interacts with substrates and products.
When the enzyme is reacting with the substrate, a complex is formed. This
interaction can be expressed as:
In the induced fit model of enzymatic activity, enzymes change shape after
binding to a substrate, improving the "fit" between the enzyme and the
substrate.
That fit between the enzyme and the substrate is responsible for lowering the
activation energy required to transform substrates into products. The graph
below illustrates the differences between the amounts of activation energy (Ea)
of an uncatalyzed reaction and the lower activation of an enzyme-catalyzed
reaction. Note that there is no difference in the final amount of free energy
( G).
Figure 2: The effect of enzyme catalysis on the energy of reaction
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND (continued)
The efficiency of an enzyme in facilitating the chemical reaction that transforms substrates to products is changed by
conditions that affect the shape of the active site. Optimal enzyme efficiency requires optimal environmental conditions,
such as pH and temperature. Raising the temperature of any substance will cause a rise in its average kinetic energy
because the heat in any given system is expressed as molecular motion. As heat is added, molecular motion increases.
As a general rule, an increase of 10 °C doubles the rate of most chemical reactions. Enzymes are proteins, however, and
so are subject to heat-induced alteration of their shape or tertiary structure. The protein has become denatured when
the tertiary structure is altered to the point that it becomes permanently inactive.
In the same manner as temperature affects enzyme activity, so does pH. Alkalinity/acidity outside the optimal range for
an enzyme will effect confomational changes in the enzyme's active site, altering its ability to interact with the substrate.
The pH range that supports an enzyme's optimal activity is associated with the natural environment in which it evolved.
The ionization state of the R groups of the amino acids that make up the protein are affected by the pH of the
environment. Therefore, the charge associated with an R group in an active site will affect how efficiently the enzyme
interacts with substrates and products. Some enzymes are not optimally active unless negative charges are neutralized
or, conversely, unless R groups are charged. Two examples are illustrated in the graph below. Pepsin is a hydrolytic
enzyme that is required to be active in the acidic environment of the stomach. Trypsin is a hydrolytic intestinal enzyme
with an optimal enzymatic activity in the slightly basic range that is produced in the pancreas
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND (continued)
Enzyme-facilitated reactions follow the Law of Mass Action: the direction taken by an enzyme-catalyzed reaction is directly
dependent on the relative concentration of enzyme, substrate, and product. An enzyme catalyzes the reaction in both
directions. When there is a great deal of substrate and little product, the reaction will form more products. Conversely,
when there is a great deal of product, the reaction may form more substrate. However, note the following two situations
where the Law of Mass Action is circumvented: (1) when the product is immediately metabolized or transported away
from the enzyme, the product concentration does not rise; (2) in highly exergonic reactions the product has little free
energy and the opposite reaction requires a large, if not unachievable, amount of energy to reverse the process.
Note that in the above graph, the initial reaction curve is very steep. As more product forms, the rate of formation levels
off as an equilibrated ratio of substrate and product is produced.
Enzymes are often tightly bound to a prosthetic group (cofactor), which is either a metal ion, an organic molecule/metal
ion complex, or a small organic molecule (coenzyme). Metal ions such as Fe+3 and Zn+2 are generally involved in reactions
which require electron removal from a substrate or which can electrically bond an enzyme to a substrate. Coenzymes
have a much more varied role: some are not tightly bound and can move from enzyme to enzyme, transferring electrons
or protons; some alter substrates to better fit with the enzyme; still others, bound into membranes, are essential to the
energy conversion reactions of photosynthesis and respiration. Many coenzymes must be taken in by animals, and are
not synthesized; these are collectively referred to as vitamins.
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND (continued)
Competitive Inhibitor
Notes:
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Molecules that bind to the active site and compete with the substrate are called
competitive inhibitors. Note that the inhibitor (its three-dimensional shape) is
similar enough to the enzyme that it can fit into the active site, but it is not the
same as the substrate. The inhibitor binds the enzyme, thus blocking substrates
from binding, and no product is produced. Competitive inhibition is reversible,
and the components behave the same as an enzyme—substrate complex, with
constant binding and unbinding of the inhibitor due to the Law of Mass Action.
If the concentration of the inhibitor is high enough, the reaction with substrate
will slow down; otherwise, the inhibitor has little effect. Increasing the
concentration of the substrate can overcome the effect of a competitive
inhibitor. The figure below illustrates the effect of a competitive inhibitor on an
enzyme. The reaction without an inhibitor proceeds very rapidly, whereas the
reaction that is competitively inhibited proceeds more slowly.
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Figure 5: Substrate Concentration
Unlike competitive inhibitors, noncompetitive inhibitors bind to a region of the
enzyme other than the active site, causing a shape change in the enzyme that
will impair the function of the active site. Since there is no competition for the
active site, the Law of Mass Action will not come into play in this situation, and
a buildup of substrate will not make a difference in accelerating the reaction.
______________________________
_
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND (continued)
Notes:
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More complex enzymes that have quaternary structures contain sites in
addition to the active sites which are called effector bonds. The effector
renders the enzyme inactive only while it is bound to the enzyme. This is a
common mechanism of control in metabolic pathways. In the pathways,
enzymes exist in two forms: active and passive. The active form is rendered
inactive by an effector, often a product of a later enzyme reaction, as illustrated
in the diagram below. When the products of a metabolic pathway inhibit an
earlier step in the pathway, it is referred to as feedback inhibition, or negative
feedback.
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Not all effectors inhibit an enzyme. In allosteric promotion, the effector
activates an inactive enzyme. Both of these regulatory mechanisms are
extremely effective and can work together, allowing the cell to store enzymes
in both their active and inactive forms. In the case of allosteric inhibition, if a
product down the metabolic line begins to build up it is not to the cell's
advantage to continue to make it. The product itself will "turn down" the
reaction until most of the product has been metabolized. In allosteric
promotion, that same product (or a different one) will activate an enzyme to
begin reaction with a substrate.
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
BACKGROUND (continued)
The Role of Turnip Peroxidase
Notes:
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Hydrogen peroxide is a highly reactive oxidizing agent that is produced in most
cells, formed spontaneously as metabolic waste. The enzyme turnip peroxidase
is found in a cell organelle called the peroxisome that serves to sequester and
break down hydrogen peroxide into relatively inert water and oxygen gas,
preventing chemical damage that would be caused by free, reactive peroxide.
Turnip cells use turnip peroxidase to break down hydrogen peroxide into water
and oxygen (Figure 8). How efficiently the enzymes are able to perform this
reaction is influenced by abiotic and biotic factors. The rate of enzymatic
activity is determined by how fast 1 unit of enzyme can convert 1μM of
hydrogen peroxide to oxygen. In this lab activity you will perform an enzymatic
activity assay in which you will assess the amount of oxygen produced by
comparing the color development of the reaction to a color palette.
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_
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
SAFETY PRECAUTIONS

As general safe laboratory practice, it is recommended that you wear proper protective equipment, such as gloves,
safety goggles, and a lab apron.

As general lab practice, read the lab through completely before starting, including any Material Safety Data Sheets
(MSDSs) and live material care sheets at the end of this booklet as well as any appropriate MSDSs for any additional substances
you would like to test. One of the best sources for the material is the vendor. For example, when purchased at Ward's, searching
for the chemical on the Ward's website will direct you to a link for the MSDS.
(Note: There are no live material care sheets included in this particular lab.)
At the end of the lab:

All laboratory bench tops should be wiped down with a 10% bleach solution or disinfectant to ensure cleanliness.

Wash your hands thoroughly with soap and water before leaving the laboratory.
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
PART 1 – STRUCTURED INQUIRY:
ESTABLISHING A BASELINE
MATERIALS NEEDED PER LAB GROUP
3

1

5

1 mL

1 mL

2 mL

4 mL


Shared Materials

2.5 mL syringes
10 mL syringe
Test tubes
Guaiacol
Turnip peroxidase solution
Dilute hydrogen peroxide
pH 7 buffer solution
Timer
1 box of Parafilm
PART 1 – PROCEDURE: STRUCTURED INQUIRY
1. Label the test tubes and syringes, as follows:
2.5 mL syringe labeled ‘E’ for enzyme – turnip peroxidase solution.
2.5 mL syringe labeled ‘P’ for product as represented by indicator. Guaiacol reacts with free O2 (product) to
form brown color.
10 mL syringe labeled ‘NB’ for neutral buffer – pH 7.
2.5 mL syringe labeled ‘S’ for substrate – 0.1% H2O2
Test tube labeled ‘A’ for mixture A
Test tube labeled ‘B’ for mixture B
2. Fill and prepare the labeled syringes and test tubes with the appropriate solutions (provided).
a. Dispense the following reagents in TUBE A:
2 mL of 'S' substrate (0.1% H2O2)
1 mL of ‘P’ indicator for product (Guaiacol -NOTE Guaiacol is an indicator for the release of oxygen, which then
turns brown)
1 mL of 'NB' neutral buffer
b. Cover tube TUBE A with parafilm and gently invert two times to mix.
c. Dispense the following reagents in TUBE B:
1 mL 'E' enzyme (turnip peroxidase)
3 mL `NB' neutral buffer
d. Cover tube TUBE B with parafilm and gently invert two times to mix.
e. Using a disposable transfer pipet, transfer the mixture from TUBE A into TUBE B.
f. Cover with parafilm and invert two times to mix.
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3. Make observations and record data.
a. Using the color palette provided by your instructor, immediately observe and compare the color of your
reaction to the color palette and record the tube # (color) of the mixture over time. This data should be
recorded in 1-minute intervals for a total incubation period of 20 minutes. INVERT THE TUBE BETWEEN
EACH READING.
 OPTIONAL: if the class has access to a probe to measure the transmittance of light or to a
spectrophotometer, more quantitative results with better resolving power can be generated by measuring
the changes in the transmittance or absorbance of light.
b. Plot the increase in color intensity (product formation) relative to your color palette over the 20-minute
interval and calculate the rate of enzymatic reaction under the baseline conditions of this experiment.
 This establishes your baseline rate.
4. Make observations and record data
 Note: The color palette represents a range of indicator color that increases 10% between tubes 1-10.
Tube 0 represents no indicator, no color.
 NOTE: The color intensity is used as a way to quantify the amount of oxygen that is produced in the
enzymatic reaction. The brown color is produced when the guaiacol reacts with oxygen (product of the
enzyme substrate reaction). Therefore, the more intense the color, the more oxygen is produced in the
reaction.
5. Set your labeled syringes aside for use in the following parts of this investigation.
NOTE: Do not throw away your labeled syringes. You will need them for the remaining parts of this
investigation
TIME INTERVAL
% TRANSMITTANCE
0
1 MIN
2 MIN
3 MIN
4 MIN
5 MIN
6 MIN
7 MIN
8 MIN
9 MIN
10 MIN
11 MIN
12 MIN
13 MIN
14 MIN
15 MIN
16 MIN
17 MIN
18 MIN
19 MIN
20 MIN
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
PART 2 – GUIDED INQUIRY: VARIABLES THAT AFFECT THE RATE OF ENZYME REACTION
MATERIALS NEEDED PER LAB GROUP






12
3
1
6 mL
6 mL
12 mL
Test tubes
2.5 mL syringes (from Part 1: ‘S’, ‘P’, ‘NB’)
10 mL syringe (Part 1)
‘P’ product guaiacol
‘E’ enzyme turnip peroxidase solution
‘S’ substrate dilute hydrogen peroxide (0.1% H2O2)
Share Materials







1
500 mL
500 mL
500 mL
500 mL
500 mL
500 mL
‘NB’ 10 mL Syringe to dispense all buffer solutions
pH 3 Buffer Solution
pH 5 Buffer Solution
pH 6 Buffer Solution
pH 7 Buffer Solution
pH 8 Buffer Solution
pH 10 Buffer Solution
PART 2 – PROCEDURE: GUIDED INQUIRY:
1. In your laboratory notebook or sheet, record the baseline rate established in Part 1.
2. Label twelve test tubes 1 through 12, respectively.
3. In each of tubes 1, 2, 4, 9, 11, and 12, dispense:
2 mL 'S' substrate dilute hydrogen peroxide using 'S' syringe
1 mL `P' product indicator guaiacol using 'P' syringe
1 mL 'NB' neutral buffer pH 7
 NOTE: All of these tubes contain the substrate.
4. In tube 3 dispense the following volumes of reagents:
1 mL of turnip peroxidase solution, using the syringe labeled 'E'
3 mL of pH 3 solution, using the rinsed 'NB' syringe
5.
In tube 5 dispense the following volumes of reagents:
1mL of turnip peroxidase solution, using the syringe labeled ‘E’
3mL of pH 5 solution, using the rinsed ‘NB’ syringe
6. In tube 6 dispense the following volumes of reagents:
1mL of turnip peroxidase solution, using the syringe labeled ‘E’
3mL of pH 6 solution, using the rinsed ‘NB’ syringe
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7. In tube 7 dispense the following volumes of reagents:
1mL of turnip peroxidase solution, using the syringe labeled ‘E’
3mL of pH 7 solution, using the rinsed ‘NB’ syringe
8. In tube 8 dispense the following volumes of reagents:
1mL of turnip peroxidase solution, using the syringe labeled ‘E’
3mL of pH 8 solution, using the rinsed ‘NB’ syringe
9.
In tube 10 dispense the following volumes of reagents:
1mL of turnip peroxidase solution, using the syringe labeled ‘E’
3mL of pH 10 solution, using the rinsed ‘NB’ syringe
10. Do not mix ALL tubes at once. You will need to take readings of each tube combination for 15 minutes each. Only
combine as many tubes that you can actually complete in this lab session!
11. Pour to combine the reagents from tube 1 with the reagents in
tube/pH 3.
To simplify the process, pair the tubes according to Table 2 below:
Table 2: Tube Pairing Chart
Tube
1
2
Tube/pH 3
5
4
6
9
7
11
8
12
10

NOTE: All of the pH tubes will
contain the respective pH buffer
solution, in case the tubes get mixed up.
12. Observe the enzyme reaction mixtures every minute for 15 minutes by comparing to the color palette (or
optional measurement of absorbance/transmittance). Record your observations in data table below.
13.
Refer back to the tube pairing chart (Table 2) and mix the remaining pairs of tubes. Repeat Step 11 for the
remaining pairs of tubes.
14.
Calculate the rate of reaction for each tube as described in Part 1. In google spreadsheets, graph your rate
results relative to pH.
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TIME
%T
TUBES 1/3
%T
TUBES 2/5
%T
TUBES 4/6
%T
TUBES 9/7
%T
TUBES 11/8
%T
TUBES 12/10
0 MIN
1 MIN
2 MIN
3 MIN
4 MIN
5 MIN
6 MIN
7 MIN
8 MIN
9 MIN
10 MIN
11 MIN
12 MIN
13 MIN
14 MIN
15 MIN
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13
PART 2 ASSESSMENT QUESTIONS
Name ______________________________
1.
Based on the graph and the overall slope of the line, what can you conclude about the effect of pH on
reaction rate? Why do you think that occurs?
2.
What happens to turnip peroxidase during and after the reaction?
3.
What would the reaction look like if you omitted parts of the reaction from the mix?
4.
What other factors may influence enzyme activity (rate of reaction)?
5.
Peroxidase breaks down hydrogen peroxide. What other types of enzymes might be needed in an
organism?
6.
You investigated peroxidase from a turnip. How might the activity of peroxidase from a mammal be
different from that of a turnip?
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
PART 3: ENZYME ACTIVITY
OPEN INQUIRY: DESIGN AN EXPERIMENT
EXPERIMENT
DESIGN TIPS
The College Board encourages peer
review of student investigations
through both formal and informal
presentation with feedback and
discussion. Assessment questions
similar to those on the AP exam might
resemble the following questions,
which also might arise in peer review:
 Explain the purpose of a
procedural step.
 Identify the independent
variables and the dependent
variables in an experiment.
 What results would you
expect to see in the control
group? The experimental
group?
 How does a specific concept
(XXXX) account for described
findings (YYYY)?
 Describe a method that could
be used to determine a given
concept/observation (XXXX).
What questions occurred to you as you completed your studies of peroxidase
activity? Now that you are familiar with enzymatic activity and ways to assess
that activity, design an experiment to investigate one of your questions.
Questions may involve assessing a range of abiotic factor effects on peroxidase
activity, modeling the kinetics of enzyme activity, comparing protein sequence
or optimal conditions for peroxidases from different sources, testing potential
chemical inhibitors of enzyme activity, comparing sensitivities of different kinds
of enzymes to the same abiotic factors, or identifying indicators for different
kinds of enzyme activity.
Before starting your experiment, plan your investigation in your lab notebook.
Have your teacher check over and initial your experiment design. Once your
design is approved, investigate your hypothesis. Be sure to record all
observations and data in your laboratory sheet or notebook.
Use the following steps when designing your experiment.
1. Define the question or testable hypothesis.
2. Describe the background information. Include previous experiments.
3. Describe the experimental design with controls, variables, and
observations.
4. Describe the possible results and how they would be interpreted.
5. List the materials and methods to be used.
6. Note potential safety issues.
After the plan is approved by your teacher:
7. The step by step procedure should be documented in the lab notebook.
This includes recording the calculations of concentrations, etc., as well as
the weights and volumes used.
8. The results should be recorded (including drawings, photos, data printouts).
9. The analysis of results should be recorded.
(continued on next page)
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AP® INVESTIGATION #13: INTERACTIONS: ENZYME ACTIVITY – STUDENT GUIDE
Kit #36W7413
PART 3: OPEN INQUIRY (CONTINUED)
Notes:
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10. Draw conclusions based on how the results compared to the
predictions.
11. Limitations of the conclusions should be discussed, including
thoughts about improving the experimental design, statistical
significance and uncontrolled variables.
12. Further study direction should be considered.
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_
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16