Lab #4 – Working with Enzymes
“How do abiotic or biotic factors influence the
Rates of enzymatic reactions? “
Objectives
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The student is able to design a plan for collecting data to show that all
biological systems are affected by complex biotic and abiotic interactions.
The student is able to use models to predict and justify that changes in the
subcomponents of a biological polymer affect the functionality of the
molecule.
The student is able to analyze data to identify how molecular interactions
affect structure and function.
The student is able to describe the role of enzymes in metabolism.
Safety
Lab-Specific Safety
 Guaiacol is a poison and flammable. Review the Material Safety Data
Sheet (MSDS) for this chemical located at the end of the lab.
General Safety
 Note where the teacher tells you to properly dispose of non-hazardous
chemicals or biological material.
 Know where all emergency equipment (safety shower, eyewash station,
fire extinguisher, fire blanket, first aid kit etc.) is located.
 Make sure to remove all dangling jewelry and tie back long hair before
they begin.
 Read all instructions, Material Safety Data Sheets (MSDSs) and live care
sheets before starting the lab activities and to ask questions about safety
and safe laboratory procedures.
 As general laboratory practice, it is recommended that students wear
proper protective equipment, such as gloves, safety goggles, and a lab
apron.
At end of lab:
 All laboratory bench tops should be wiped down with a 20% bleach
solution or disinfectant to ensure cleanliness.
 Wash their hands thoroughly with soap and water before leaving the
laboratory.
Materials
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Read the procedures and construct a list of materials for the lab exercises.
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Background
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Read the background information and answer the following questions
under background section of your lab notebook:
o Give the typical enzyme reaction. Label the enzyme, substrate,
enzyme-substrate complex and product. Explain the process.
o Draw a graph depicting two chemical reactions, one without
enzyme and with an enzyme. Label your axis, activation energy,
and lines with a key.
o Describe how pH and temperature change a chemical reaction
facilitated by an enzyme.
o Describe how the Law of Mass action relates to amount of enzyme,
substrate and product in the chemical reaction.
o Define the following terms: coenzyme, cofactor, competitive
inhibitor, noncompetitive inhibitor
o Give the equation for the reaction being demonstrated in this lab
investigation. Identify the name of the enzyme, substrate and
product.
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:
Figure 1
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
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lower activation of an enzyme-catalyzed reaction. Note that there is no
difference in the final amount of free energy (Δ G).
Figure 2
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. The more heat that is
added, the more molecular motion will occur. 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 so much
that it becomes permanently inactive.
The relative acidity or alkalinity of an enzyme’s surroundings also influences its
activity through a similar mechanism as temperature
– by altering the enzyme’s interaction with substrates through conformational
changes in the active site. 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 will not be optimally active until
negative charges are neutralized or, conversely, until R groups become charged.
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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
hydrolytic intestinal enzyme with an optimal enzymatic activity in the slightly basic
range that is produced in the pancreas.
Figure 3
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. 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, here are two situations where the Law of Mass Action is circumvented.
When the product is immediately metabolized or transported away from the
enzyme, the product concentration does not rise. 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.
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Note that in the above graph, the initial reaction 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
Figure 4 molecule (coenzyme). Metal
ion complex, or a small organic
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.
Competitive Inhibitors
Molecules that bind to the active site and compete with the substrate are called
competitive inhibitors. Note that the inhibitor is similar enough to the enzyme that
it can fit in the active site, but it is not the same as the substrate. The inhibitor
binds the enzyme, therefore blocking substrates from binding, and no product is
produced. Competitive inhibition is reversible and behaves 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 large
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
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.
Figure 6
The Role of Turnip Peroxidase
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 7). How efficiently the enzymes are able 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.
Turnip Peroxidase
2H202

2H20 + O2
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Figure 7
Procedure
Part 1: Establishing a Baseline
Materials
1 mL syringes
2.5 mL syringe
10 mL syringe
15 x 125 mm tubes
Disposable 9” transfer pipet
Guaiacol
Turnip peroxidase solution
Dilute hydrogen peroxide
pH 7 buffer solution
Timer
Parafilm
Procedure Steps
1. Label the test tubes and syringes, as follows:
a. 2.5 mL syringe labeled “E” for enzyme- turnip peroxidase
solution
b. 2.5 mL syringe labeled “P” for product as represented by the
indicator Guaiacol. Guaiacol reacts with free O2 (product) to
form brown color.
c. 10 mL syringe labeled “NB” for neutral buffer – pH 7
d. 2.5 mL syringe labeled “S” for substrate – 0.1% H2O2
e. Test tube labeled “SPNB” for mixture A
f. Test tube labeled “ENB” for mixture B
2. Fill and prepare the labeled syringes and test tubes with appropriate
solutions.
a. Dispense the following reagents in “Tube SPNB”:
i. 2 mL of S substrate
ii. 1 mL of P product
iii. 1 mL of NB neutral buffer
b. Cover the tube with parafilm and invert two times to mix.
c. Dispense the following reagents in “Tube ENB”:
i. 1 mL of E enzyme
ii. 3 mL of NB neutral buffer
d. Cover the tube with parafilm and invert two times to mix.
e. Using a disposable pipette, transfer the mixture from tube
“SPNB” to tube “ENB.”
f. Cover with parafilm and invert two times to mix.
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3. Make observations and record data.
a. In your lab notebook in the data section, title this Part 1:
Establishing a Baseline. Under the title construct a data table
with seven columns and two rows. This is Table 1. 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 five minutes.
b. Under table 1, construct a graph. Plot the increase in color
intensity (product formation) relative to your color palette over
the 5-minute interval and calculate the rate of enzymatic
reaction under the baseline conditions of this experiment.
Note: The color palette represents a range of indicator color that
increases 10% between tubes 1-10. Tube 0 represents no indicator, no
color. The color intensity is used as 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.
4. Set your labeled syringes aside for use in the following parts of this
investigation.
Part 2: Variables that affect the Rates of Enzyme Reactions
Materials
15 x 125 mm tubes
2.5 mL syringes (from Part 1)
10 mL syringe (from Part 1)
‘P’ product guaiacol
‘E’ enzyme turnip peroxidase solution
‘S’ substrate dilute hydrogen peroxide (0.1% )
‘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
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Procedure Steps
1. In your laboratory notebook, title this section Part 2: Variables that affect
the Rates of Enzyme Reactions.
2. Write the question being proposed by this part of the lab. Underneath the
question identify the independent variable, the dependent variable and
three standardized variables in this investigation.
3. Under the variables, title and record the baseline rate from Part 1.
4. Next, construct a data table that will allow you to record the color of the
seven different pH test tubes over a period of 5 minutes record at oneminute intervals. This is Table 2.
5. Label twelve 13 x 100 mm tubes 1 through 12, respectively.
To simplify the process, pair the tubes according to the chart below:
Tube
Tube/pH
1
3
2
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.
6. 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.
7. 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
8. Repeat step 4 for tubes 5, 6, 7, 8, and 10 with their respective pH buffers.
9. Using a disposable 9” transfer pipette, combine the reagents from tube 1
with the reagents in tube/pH 3.
10. Observe the enzyme reaction mixtures every minute for 5 minutes by
comparing to the color palette. Record your observations in your
laboratory notebook in table 2.
11. Refer back to the tube pairing chart and mix the remaining pairs of tubes.
Repeat Steps 6 and 7 for the remaining pairs of tubes.
12. Calculate the rate of reaction for each tube as described in Part 1. In your
laboratory notebook, construct a graph under your table 2 expressing your
rate results relative to pH.
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Assessment Questions
Answer the following questions in your lab notebook in complete sentences.
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 left parts of the reaction out of 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?
Part 3: Enzyme Activity- Open Inquiry: Design an Experiment
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 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:
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7. The step by step procedure should be documented in the
lab notebook.
Writing About Your Results
From the data that you collected from your independent investigation in
Procedure 3, write a report that follows the guidelines below:
Include:
1. A Title that communicates the question being answered in the
paper.
2. An Introduction that provides the context of your investigation and
states the question asked and the hypothesis tested in the study.
3. A Materials and Methods section that describes your experiment
in such away that it can be repeated. This should be written as a
narrative description that integrates the materials with the
procedures used in the investigation.
4. A Results section that consists of
1. One or two sentences about the nature of the research
2. One or more paragraphs that describe the results
3. One or more tables showing the data collected during the
investigation
4. One or more graphs displaying trends or comparisons that
help the reader understand your findings
5. A Discussion section in which you analyze and interpret the results
of your experiment. What do your results tell you about the effect of
a biotic and/or an abiotic environmental factors on the rate of
enzymatic reactions?
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Pre- Lab Set- Up for Lab Notebook
Complete the following sections for your notebook before the
lab begins. Be sure to leave plenty of space to complete the
different parts, graphs and questions. Make titles bold and
stand out. Be neat and organized.
I.
II.
III.
IV.
Objective(s)
Safety
Materials – use pages 7-8
Background – Read pages 2-6. Write out
questions on page 2 and answer in complete
sentences.
V. Procedure – write out the detailed procedure
steps for Part 1 (pages 7-8) and Part 2 (page 9).
Be sure to write in past tense and in third
person.
VI. Data – Complete the following:
1. Part 1 – read step 3a on page 8. Complete
your title and Table 1. Leave room for your
graph by drawing an X and Y axis (preparing
for construction of the graph.)
2. Part 2 – read steps 1 and 2 on page 9.
Complete your title, question and identify
your variables. Then read step 3 and leave a
space for baseline information. Finally read
step 4 and draw your Table 2. Draw your X
and Y axis for your second graph. Leave
room!
VII. Assessment Questions - On page 10 write out
the questions leaving room for you to answer
them.
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