Enzymes and Their Functions

Authors: Jeisa Pelet and Carolyn Wilczynski

Date Created: 2009-2010
Subject: Living Environment
Level: High School
Standards: New York Sate Learning Standards Living Environment
(http://www.emsc.nysed.gov/ciai/)
Standards 1: Analysis, Inquiry and Design
Standards 4: Physical Setting and Living Environment
Schedule: 5 periods of 45-minutes and 2 periods of 90-minutes

Objectives:
Students will learn about enzymes and
how their activities are affected by
different environmental factors.
Following an introductory activity,
students will conduct an experiment
using amylase (enzyme) and starch
(substrate) as an example.
Students will:
Understand the concept of enzymes,
chemical reactions, catalysts and
substrates.
Correlate locks and keys with
enzymes and substrates.
Quantify the production of glucose
from amylase/starch reaction in time.
Develop an experimental design
based on different factors affecting
enzyme activity.
Develop a problem statement and
hypothesis, collect and analyze data,
arrive at conclusions and complete a
lab report.

Vocabulary:

Enzymes
Chemical reaction


Catalyst
Substrate

Materials:
For Each Pair:
Part 1:
Locks and keys
Part 2 and 3 (each):
600 mL Beaker
Dialysis tubing (~3cm
width, molecular weight
cutoff of 12,000 to 14,000
MW, Carolina) (15 cm)
Starch (3 g)
Amylase (1 g)
Benedicts solution (3 mL)
Pipettor (P1000)
Pipette tips (1 mL)
Transfer pipettes (2)
2 mL Eppendorf tubes (7)
Floating eppendorf tube
holder

Tube holder
Plastic cuvettes (7)
Cuvette holder
Waste container
Magnetic stir bar
(3.5-5 cm)
Dialysis clips (2)
Timer
Stir plate
Stir/heating plate

For Each Student:
Safety goggles
Gloves
Activity sheets

For the Class:
Spectrophotometer
Small centrifuge
Vortex
Safety:
Benedicts solution should be handled with care.
Students should wear safety goggles,
lab coats, and gloves when working with Benedicts
solution and hot water. If possible, use heat
resistant gloves when handling hot water.


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Introduction

To study how the enzymes act upon the substrates, we will use amylase
and starch as an enzyme and a substrate, respectively. Amylase converts
polysaccharides into monosaccharides. Polysaccharides are long chains of
sugars attached together, while monosaccharides are single sugar molecules. In
the case of starch (a polysaccharide), amylase will break it down into glucose
(monosaccharide) as illustrated below.






If amylase and starch are placed inside a dialysis tubing, the amylase will
immediately begin to break the starch down into glucose. As the glucose forms
from the enzyme activity, it will diffuse out through the membrane because it is
small enough to fit through the mesh of the membrane. However, the amylase
and the starch will stay inside the dialysis tubing because these molecules are
too large to fit through the mesh. By detecting the amount of glucose outside the
dialysis tubing over a period of time, we can study the rate (how fast or how slow)
of the enzyme activity.

This lesson on enzymes consists of three parts:

Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity
Part 2 (Day 3 4): Enzyme Action: The Breakdown of Starch into Glucose
Part 3 (Day 5 7): Inquiry-based Activity on Enzymes

All three parts can be performed sequentially. However, if time is a constraint,
each part can be done independently, except for Part 3 which requires Part 2 as
an introduction.

Students Pre-requisites:

It is recommended that the following Lab is completed before this lesson:
New York State Education Department (NYSED) . 2002. "Diffusion Through a
Membrane". A laboratory activity produced by the State Education Department
for use in fulfilling part of the laboratory requirement for the Regents Examination
in Living Environment (www.nysed.gov).

If the 'Diffusion Through a Membrane' lab is not available, the main concepts
covered by this lab can be found in the Living Environment Core Curriculum:
http://www.emsc.nysed.gov/ciai/mst/pub/livingen.pdf
amylase
starch glucose


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Science Content:

What are Enzymes?

Enzymes are compounds that assist chemical reactions by increasing the
rate at which they occur. For example, the food that you eat is broken down by
digestive enzymes into tiny pieces that are small enough to travel through your
blood stream and enter cells. Enzymes are proteins that are found in all living
organisms. Without enzymes, most chemicals reactions within cells would occur
so slowly that cells would not be able to work properly. Enzymes function as
catalysts. Catalysts accelerate the rate of a chemical reaction without being
destroyed or changed. They can be reused for the same chemical reaction over
and over, just like a key can be reused to open a door many times. Enzymes are
generally named after the substrate affected, and their names usually end in -
ase. For example, enzymes that break down proteins are called proteases.
While lipases break down lipids, carbohydrases break down carbohydrates.

The compounds that enzymes act upon are known as substrates. The
substrate can bind to a specific place in the enzyme called the active site. By
temporarily binding to the substrate, an enzyme can lower the energy needed for
a reaction to occur, thus making this reaction faster. The energy required for a
chemical reaction to occur is known as the activation energy. Once the reaction
between an enzyme and a substrate is complete, the substrate is changed to a
product while the enzyme remains unchanged. The rate of the reaction between
an enzyme and a substrate can be affected by different factors. Some of the
factors that can affect enzyme activity are temperature, pH, concentration of the
enzyme and concentration of the substrate. In living organisms, enzymes work
best at certain temperatures and pH values depending on the type of enzyme.


A Little More Information on Diffusion and Dialysis Membranes

Molecules are in constant motion. So when there is a difference in
concentration, or a concentration gradient of a specific particle, the particles will
move toward the lower concentration in order to maintain an evenly distribution
across the whole area. This movement of particles from higher concentration to
lower concentration is known as diffusion.
A dialysis membrane is a mesh-like material that will only allow certain
particles of a specific size or smaller to pass or diffuse through. For example, if
inside a dialysis tubing we place grains of rice and salt, the salt will diffuse out
the tubing because it is small enough to move through the mesh of the
membrane. The rice, however, will stay inside the tubing because it is too large
to move through the mesh of the membrane. Kidneys are similar to a dialysis
membrane. As blood passes through the kidneys, small molecules will be filtered
out and eliminated from the body as urine.


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Preparation:

1. Photocopy the Activity Sheets corresponding to each Part (per student).
2. Perform the following preparation steps for each Part accordingly.

Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity

Prepare two individual sets of 4 locks and 4 keys each (one per group).
Set 1: Locks and keys will all have different shapes and sizes, and each key
will open only one lock.
Set 2: Locks and keys will all have different shapes and sizes, one key will
open two locks, one key will open only one lock and two keys will not open
any locks.


Part 2 (Day 3 5): Enzyme Action: The Breakdown of Starch into Glucose

A. Glucose calibration curve
A calibration curve of glucose concentration with absorbance should be
performed. This calibration curve is done by preparing various solutions of
different known concentrations of glucose with fixed amount of Benedicts
solution. The absorbance of each sample is then detected using a
spectrophotometer, and a plot of absorbance (y-axis) vs. glucose concentration
(x-axis) is generated. A linear regression of this data will provide a slope (m) and
a y-intercept (b) values according to the following equation:
y = (m x) +b
The m and b values will be used for the calculation of glucose
concentration in the unknown samples. Detailed instructions on how to perform
the calibration curve can be found in Supplementary Information.
Alternatively, if time is a constraint, the values of m = -0.81 and b = 0.59 can
be used.

B. Materials to be prepared prior to lab day

1. In a vial, weigh 3g of starch (one per group).
2. In a vial, weigh 1g of amylase (one per group).
Note: If amylase is not going to be used immediately, place in the fridge after
weighing and do not dissolve with water until ready for the experiment.
3. Cut 15 cm of dialysis tubing (one per group).

Part 3 (Day 4 7): Inquiry-Based Activity on Enzymes

Part 3 requires the same materials and preparations as in Part 2.
Additional materials (i.e. pH meter, sodium chloride (NaCl) solution, sodium
hydroxide (NaOH) solution, ice) might be required depending on the experiment
designed by the students.


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Classroom Procedure:

Part 1 (Day 1 2): Enzymes and Their Functions Lock-and-Key Activity

Objective: The objective of this activity is to introduce the concept of enzymes
and their functions through the lock-and-key model by using real locks and keys
as an analogy. Students can be divided in groups of 2 or 3.

Procedure Part 1.1:
1. Hand out the Activity Sheets Part 1 (page 3 7) (per student).
2. To each group of students, provide Set 1 of locks/keys.
3. Ask each group to try all the keys with all the locks and to answer the
following questions:
a. Were you able to open all locks?
b. Do all keys open all locks?
c. Can 1 key open more than 1 lock?
d. Can you open the same lock with the same key more than once?
e. Do all keys have the same shape?
4. Collect Set 1 of locks/keys and continue with Part 1.2.

Procedure Part 1.2:
1. Ask each group of students to make 3 predictions (from previous
observations) about a new set of locks/keys to be given (Set 2).
2. To each group provide Set 2 of locks/keys.
3. Ask the students to test their predictions and to say if whether or not each
prediction was valid based on their results.
4. Ask the students to make 3 additional observations about Set 2 of locks/keys.
5. Ask the students to make comparisons between enzymes/substrates and
keys/locks (3 similarities and 3 differences).
6. Ask the students to share their ideas and write them on the blackboard.
7. Allow students to complete the questions in the Activity Sheets Part 1 (B).

After this activity, the lock-and-key model can be correlated to enzymes
and substrates. Different properties on enzymes/substrates can be
addressed and compared to keys/locks. For example, enzymes (keys) are
reusable, specific, have different shapes, produce a change to a substrate
(lock), and fit in an active site with a substrate. It would also be important
to talk about factors that affect enzyme activity, such as temperature, pH
and enzyme/substrate concentration.



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Part 2 (Day 3 4): Enzyme Action: The Breakdown of Starch into Glucose

Objective: The objective of this activity is to perform an experiment with an
enzyme (amylase) and a substrate (starch), and to understand how enzymes
work. Each group of students will set up the same control experiment (6 time
points) for 30-40 minutes (depending on the time available). The amount of
glucose produced from the amylase/starch reaction will be quantified in time.
Data will be compared among students. Students should be divided in groups of
2 or 3.

Procedure Part 2.1: Collecting Samples (Day 3)
1. Provide the following materials to students (per group):
a. Beaker (600mL)
b. Stir plate
c. Stir bar
d. Graduated cylinder (500 mL)
e. 15 cm Dialysis tubing (pre-cut)
f. Pre-weighted starch (3g)
g. Pre-weighted amylase (1g)
h. Water (~510 mL)
i. Pippetor (P1000)
j. Pipette tips (1 mL)
k. Waste container
l. Transfer pipette (2)
m. 2 mL Eppendorf tubes (7)
n. Tube holder
o. Timer
p. Vortex (1 per class)
q. Activity Sheets Part 2 (page 8-14)
(per student)

The following are the steps for collecting samples (same as in Activity
Sheets, Page 10):
2. Select the times at which samples will be collected.
Note: Recommended times are 0, 5, 10, 20,30 and 40 minutes.
3. Write these times (minutes) in Table 1.
4. Label 7 eppendorf tubes with numbers from 1 to 7 and your initials.
5. Using a graduated cylinder, measure 500 mL of water and place it in the
beaker.
6. Remove 1.5 mL of the water from the beaker and place it in tube 1.
7. Dissolve 3 g of starch by adding 5 mL of water to the vial.
Note: a white cloudy solution should be seen.
8. Shake or vortex the starch solution.
9. Dissolve 1 g of amylase by adding 5 mL of water to the vial.
Note: A brown solution should be seen.
10. Shake or vortex the amylase solution.
11. Seal the bottom of the dialysis tubing by wrapping one of the ends around a
stir bar and clipping this end with a dialysis clip.
12. Use a transfer pipette to add all of the starch solution into the dialysis tubing.


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13. Use another transfer pipette to add all of the amylase solution into the dialysis
tubing.
14. Clip the other end (top part) of the dialysis tubing with a dialysis clip.
15. Insert the sealed dialysis tubing in the beaker with water.
16. Start the timer.
17. Set the stir dial in the stir plate to the lowest setting.
18. At each specific time (see Table 1), remove 1.5mL of the water from the
beaker and place it in a 2 mL eppendorf tube labeled with the number
corresponding to that time (tubes 2 6).
19. To tube 7, add 1.5 mL of water.

Additional Notes:
Note 1: if pippettors are not available, use graduated plastic pipettes to
collect the samples.
Note 2: The amylase will form a brown layer on the top while the starch will
form a white layer on the bottom of the dialysis tubing. As time passes, the
brown layer (amylase) will increase as the white layer (starch) will decrease.
Note 3: If the data analysis is going to be done on a different day, the
samples collected by the students can be stored in the freezer.
Note 4: A negative control experiment can be performed where only starch is
placed in the dialysis tubing (no amylase) and Benedicts solution is used to
detect for the presence of glucose after 30-40 minutes.

Procedure Part 2.2: Analyzi ng the Data (Day 4)
1. Provide the following materials to students (per group):
a. Beaker (600 mL)
b. Stir/heating plate
c. Stir bar
d. Samples from Part 2.1
e. Benedicts solution (~2 mL)
f. Water (~300 mL)
g. Pippettor (P1000)
h. Pipette tips (1 mL)
i. Waste container
j. Plastic cuvettes (7)
k. Cuvette holder
l. Floating eppendorf tube holder
m. Timer
n. Small centrifuge (1 per class)
o. Vortex (1 per class)
p. Spectrophotometer (1per class)

The following are the steps for analyzing the data (same as in Activity
Sheets, Page 11):
2. Start a stirring water bath at ~80 90C using a heating/stir plate.
Note: Do not allow water to boil.
3. Turn on the spectrophotometer.
Note: General instructions on how to use a spectrophotometer are in
Supplementary Information.


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4. Label 7 plastic cuvettes with numbers from 1 to 7.
5. To each sample (1 6), except tube 7, add 0.3 mL of Benedicts solution.
Note: tube 7 will have only water.
6. Shake or vortex each tube for 5 seconds.
7. Place all the tubes in a floating eppendorf tube holder.
8. Place the floating eppendorf tube holder in the heating water bath.
9. Start the timer.
10. Place a stir bar in the water bath and set the stir dial in the plate to the lowest
setting.
11. After 10 minutes, remove the samples from the water bath.
Note: Caution! Floating eppendorf tube holder and tubes will be very hot.
Remove the tube holder using the tab that is above the water level. If
possible, use heat resistant gloves.
12. Allow the samples to cool down to room temperature (~3 minutes).
13. Centrifuge the tubes for 3 minutes.
14. Gently remove your samples from the centrifuge and place them in the tube
rack. Do not shake them.
15. Without disturbing the solution in your sample, use a pippetor to remove 1 mL
of the liquid and place it in the plastic cuvette with the same number.
Note: Do not to touch the bottom of the tube where the red precipitate may be
present.
16. Set the spectrophotometer to 735 nm.
17. Use cuvette 7 (only water) to set a reference for the spectrophotometer.
18. Obtain the absorbance of each sample (cuvettes 1 6).
19. Write down the absorbance of each sample in Table 1.
20. Obtain the m and b values from the glucose calibration curve.
21. Calculate the glucose concentration for each sample, and complete Table 2.
Note: Glucose concentration is calculated with: x =
-b
m
, where y is the
absorbance, and m and b are from the glucose calibration curve.
22. With the data in Table 2, plot glucose concentration (y-axis) vs. time (x-axis).
The following plot of glucose concentration vs. time should be expected:

0
0.05
0.1
0.15
0.2
0 20 40 60
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t
r
a
t
i
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(
m
g
/
m
L
)
Ti me (mi nutes)
Glucose Concentration Vs. Time


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Part 3 (Day 5 7): Inquiry-Based Activity on Enzymes

Objective: The objective of this activity is to understand how different factors can
affect enzyme activity. Each group of students will select an environmental factor
that they would like to analyze and they will design their own experiment using
the knowledge gained from the previous activities. This experiment will answer a
question based on the effect of the selected factor on enzyme activity. Students
can be divided in groups of 2 or 3.

Procedure Part 3.1: Designing an Experiment (Day 5)
1. Hand out Activity Sheets Part 3 (page 14 20) (per student).
2. Ask the students to write down in the Lab Report two factors that they think will
affect enzyme activity.
3. Allow the students to discuss these factors among their group and to pick one
factor to study.
4. Allow the students to design an experiment with their partner(s) that will
answer a question based on the selected factor and enzyme activity, and to
complete Part A and B of their Lab Report.
Note 1: Examples of factors can be: temperature (higher or lower), pH (acid or
basic) or enzyme/substrate concentration. (Make sure that you approve the
experiment to be performed). Students can follow the basic procedure in Part
2.1 and 2.2 with adjustments depending on their experimental design.

Procedure Part 3.2: Inquiry-Based Activity (Day 6 7)
1. Provide materials to students. This will depend on their experimental design
(basic materials are those from Part 2.1 and 2.2).
2. Allow the students to set up and run their experiments, collect data, discuss
their results and complete the rest of the Lab Report.
Note: Students should use the data from Part 2 to compare to their results.
An example of temperature effect on enzyme activity is as following:

0
0.2
0.4
0.6
0.8
0 20 40 60
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C
o
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t
r
a
t
i
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(
m
g
/
m
L
)
Ti me (mi nutes)
Glucose Concentration Vs. Time (T=48C)


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Supplemental Information:

Glucose Calibration Curve:

Materials:
a. Stir plate
b. Stir/heating plate
c. Glucose (2 mg)
d. Water
e. Benedicts solution
f. Pipettor (P1000)
g. Pipette tips (1 mL)
h. 2 mL Eppendorf tubes (8)
i. Tube holder
j. Plastic cuvettes (8)
k. Cuvette holder
l. Floating eppendorf tube holder
m. Vortex
n. Small centrifuge
o. Spectrophotometer
Procedure:
1. Start a stirring water bath at ~80-90C using a heating/stir plate.
Note: Do not allow water to boil.
2. Turn on the spectrophotometer.
3. Weigh 2 mg of glucose and place it in an eppendorf tube or vial.
4. Dissolve the glucose with 2 mL of water to make a 1 mg/mL solution.
5. Label 8 eppendorf tubes (2 mL) with numbers from 1 to 8.
6. Add the following (from Table S1) to each tube with the corresponding
number:
Table S1
Tube # Glucose stock
solution, 1 mg/mL
(mL)
Distilled
water (mL)
Benedicts
solution
(mL)
Final
concentration
(mg/mL)
1 0 1.50 0.3 0.00
2 0.075 1.425 0.3 0.05
3 0.15 1.35 0.3 0.10
4 0.25 1.25 0.3 0.17
5 0.5 1.0 0.3 0.33
6 0.75 0.75 0.3 0.50
7 1 0.50 0.3 0.67
8 0 1.80 0 0.00

7. Vortex each tube for 5 seconds.
8. Place all the tubes in a floating eppendorf tube holder.
9. Place the floating eppendorf tube holder in the heating water bath for 10
minutes.


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10. Remove the tubes from the water bath and allow them to cool down to room
temperature (~3 minutes).
11. Centrifuge the tubes for 3 minutes.
12. Without disturbing the solution in your sample, use a pippetor to remove 1mL
of the liquid and place it in the plastic cuvette labeled with the same number.
Note: Do not to touch the bottom of the tube where the red precipitate may be
present.
13. Set the spectrophotometer to 735 nm.
14. Use cuvette 8 (only water) to set a reference for the spectrophotometer.
15. Obtain the absorbance of each sample (cuvettes 1 7).
16. Record the data in Table S2, and plot absorbance (y-axis) vs. glucose
concentration (x-axis).
Note: a linear plot with a negative slope should be observed.
17. Perform a linear trend (in excel) and obtain the slope (m) and y-intercept (b)
values according to the following equation: y = (m x) +b

Table S2
Tube # Final glucose concentration
(mg/mL)
Absorbance at 735 nm
1 0.00
2 0.05
3 0.10
4 0.17
5 0.33
6 0.50
7 0.67

Slope (m) =_______________________________
y-intercept (b) =____________________________
The following plot of glucose concentration with absorbance is expected
(in this case, galactose was used as the monosaccharide):

y=0.8125x+0.5922
R=0.9964
-0.1
6E-16
0.1
0.2
0.3
0.4
0.5
0.6
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70
A
b
s
o
r
b
a
n
c
e

(
7
3
5

n
m
)
[Gal actose] (mg/mL)
Calibration Curve


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Instructions for using the Spectrophotometer:
For Spectro Master (Model 415) spectrophotometer (other models may vary)

1. Turn on the spectrophotometer. Allow to warm for ~30 minutes.
2. Open the sample compartment and set the filter selector to the appropriate
position depending on the desired wavelength range.
3. Use the dial on the right side to set the wavelength to 735 nm (shown in the
front window).
4. Press Trans/Abs to select ABS in the front panel.
5. Insert cuvette with only water into the holder inside the sample compartment.
The smooth side of the cuvette should be facing forward.
6. Close the lid.
7. Press 100%/T/0 A and wait for a reading to appear in the front panel. This
reading should be 0.000.
8. Remove the cuvette from the holder inside the sample compartment and
place the new cuvette into this holder.
9. Close the lid and wait for a reading to automatically appear in the front panel.


Basic Materials for Part 2 and Part 3:







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Safety:

Benedicts solution should be handled with care. It is advised that students
protect their eyes and skin by wearing protecting goggles and gloves when
working with Benedicts solution and hot water. If possible, use heat resistant
gloves when working with hot water.


Acknowledgements:

Glucose quantification was adapted from the Cell Biology Technique Manual:
Benedicts Test (Hendrix College Biology Department).
http://www2.hendrix.edu/biology/CellWeb/Techniques/Benedicts%20Test.htm

Cornell BME NSF GK-12 program: DGE 0841291.

Thanks to Dr. Michael Shuler, Dr. Chris B. Schaffer, Dr. Shivaun Archer,
Nevjinder Singhota and Dr. David Putnam. Cornell University, Ithaca NY