LAB 5

Enzyme Activity
Big Idea 4: Interactions

How do abiotic or biotic factors influence the rates of enzymatic reactions?

Please be sure you have read the

student intro packet before you do this lab.

(If needed, the student intro packet is available at www.qualitysciencelabs.com/AdvancedBioIntro.pdf)

Lab Investigations Summary

Pre-lab Questions

Lab Investigation 5.1: Enzyme Specificity

Lab Investigation 5.2: Enzyme Rate of Reaction Assay
and Baseline
Lab Investigation 5.3

Part 1 - Determining the Effect of pH on Enzymatic Activity

Part 2 - Student Guided Inquiry

Student Guided Inquiry of the Environmental Effects on Enzyme
Activity

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Lab 5 - Enzyme Activity

Big Idea 4: Interactions
How do abiotic and biotic factors influence the rates of enzymatic reactions?

BACKGROUND
Of the 30,000 proteins that DNA codes for, many are individual specific enzymes
designed for driving specific reactions. Proteins are not just long chains of amino
acids. They are complex, 3D structures that have their folded shape determined by
the sequencing of the amino acids. One incorrect amino acid (or one nucleotide
in the codon of three amino acids) in a sequence of 600 amino acids (equivalent to
1800 nucleotides) can change the 3D configuration and render the protein enzyme
useless. Enzyme specificity is built into every enzyme structure.

Figure 5.0a

Activation Energy
with Enzyme
Overall Energy

Energy

Activation Energy
without Enzyme

Progress of Reaction

How do enzymes work? Enzymes act as catalysts. They lower the activation
energy needed to drive a reaction.
Enzymes are biological catalysts. In their large folded configuration, they
have very specifically shaped substrate binding sites. These binding sites make
substrates go into the transition state (the peak of the activation energy curve).
There is an intermediate or transitional product between reactants and final
products. To catalyze the reaction, several regions of the binding site must be
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precisely positioned around the substrate molecules, creating active sites. This is
similar to a key fitting in a lock. When the enzyme attaches itself to the substrate
molecule, a new temporary transitional molecule is formed, called the enzymesubstrate complex. Within this complex, the bonds that need to be broken or
formed in the reaction are in close proximity to the active site.
Substrate

Binding Pocket

Enzyme-Subrstrate
Complex

Figure 5.0b

Products

Catalytic Site

Enzyme

Because the bond between the enzymes active site and substrate causes
intermolecular bond strain due to electromagnetic interactions, the bond breaking
or forming of the substrate requires less activation energy. Figure 5.0c shows the
breakdown of a large molecule into two smaller products with the use of an enzyme.
All chemical reactions in biological systems require the presence of enzymes.
If the 3D configuration of the protein enzyme is changed in any way, the shape of
the binding site could be affected and the enzyme becomes nonfunctional.

How Do Biotic and Abiotic Factors Affect Enzyme Activity?

pH

The optimal pH conditions are different for each enzyme. Some enzymes
Figure 5.0c
function better in acid environments.
These might be found in the stomach
involved in digestion (like pepsin at
pH of 1.5-2, a protease enzyme that
breaks peptide bonds in proteins). The
main enzyme for this lab is lactase
which decomposes the disaccharide
sugar lactose (milk sugar) into
L ac
ta se
two monosaccharides: glucose and
galactose.
Glucose
(Have you noticed that sugars
Lactose + Water
end in ose and enzymes end in
ase?) There are different lactase
enzymes that function in different
pH environments. Some are more
active in an acidic pH range from 4.0
to 5.0 and some are more active in a
Galactose
neutral pH of 7.0.
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Temperature
Temperature is an abiotic condition, and similar to pH, enzymes have optimal
temperatures at which they function best, which is usually the average temperature
of the organism. Temperature can affect the enzyme by altering its shape or even
breaking bonds. Higher temperatures can denature the enzyme protein, rendering
the enzyme irreversibly nonfunctional. Many proteins are denatured around 40 C
to 50 C, but some are still active at 70 C to 80 C, and a few even withstand
boiling. If the temperature is much lower, the reaction usually goes at a slower rate.
Substrate and Enzyme Concentrations
The concentration of both substrates and enzymes influence the rate of enzymatic
reaction. As the enzyme concentration is increased, there is an increase in product.
If there is a decrease in enzyme concentration, the rate at which the products are
formed is also decreased. Enzymes can only attach to one substrate and form an
ESC one at a time; so the more enzymes, the faster the reaction. When saturation
occurs, all of the substrate has reacted with all of the enzymes, and the reaction
stops, unless more substrate is provided.

What Other Cellular Processes Control Enzymes and

Affect Enzyme Activity?

Enzymatic Competition
Enzymatic competition occurs when there are several different enzymes
available to combine with a given substrate. Because enzymes compete with a
specific substrate site, each has fewer changes to interact with the substrate.
Negative Feedback
Negative feedback controls protein synthesis in the body. When the concentration
of products reaches a certain level, production ceases. This prevents too much
synthesis of a product. This is one example of homeostasis at work in your body.
Activators and Inhibitors
There are many molecules other than the substrate which may interact with an
enzyme. If a molecule increases the rate of reaction it is an activator. An inhibitor
is a molecule that binds to the enzyme and interferes with the formation of the
ESC (Enzyme-Substrate Complex), so the reaction cannot occur. Some examples
of inhibitors are detergents or organic solvents which tend to unfold the enzyme.
Many inhibitors react with the enzymes side chains in or near the active site to
change its shape or block it; such as poisons like cyanide and curare.

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How Are Enzymes Classified?

Enzymatic proteins 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- from the Greek to break down) and
anabolic enzymes (a- or an- from the Greek anabole, meaning to build up).
Catalytic enzymes that break down proteins are called proteases and are found
in many organisms. One example is bromelain, which comes from pineapples.
Ever wonder why pineapple cannot be added to Jell-O? The gelatin stays liquid
and will not set. The bromelain enzyme breaks down gelatin and is commonly used
in meat marinades to break down protein in meats. 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), but
they can take supplements containing lactase, the enzyme they are missing. All of
these enzymes hydrolyze large complex molecules into their simpler components.
Hydrolysis is the process of adding water and bonds are broken, whether sugars,
proteins, or nucleotides, (in the case of ATP to ADP).
Anabolic enzymes are dehydration processes that remove water and build bonds.
They build larger molecules from smaller parts. Have you heard of the controversial
anabolic steroids for muscle-building? Another example of an anabolic enzyme is
ATP synthase, the enzyme that stores cellular energy in ATP by combining ADP
and phosphate. These are kinase enzymes that phosphorylate or add phosphate
groups. The DNA and RNA polymerase enzymes involved in DNA replication
and RNA protein synthesis are also anabolic, building enzymes.
One more example of an anabolic enzyme is Ribulose-1,5-bisphosphate
carboxylase oxygenase, most commonly known by its nickname RuBisCo. It is an
enzyme involved in the Calvin cycle that catalyzes the first major step of carbon
fixation, a process by which the atoms of atmospheric carbon dioxide are made
available to organisms in the form of energy-rich molecules such as glucose.

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Preview of Enzyme Activity Lab Investigations:

The enzymes lab investigations all use the milk sugar (lactose) enzyme lactase to
explore:
Specificity (can lactase also decompose another disaccharide besides lactose sucrose?) in Lab Investigation 5.1
How to measure the rate of reaction of an enzyme and how to use a base line
assay if needed in Lab Investigation 5.2;
How to determine the effect of pH on enzyme activity in Part 1 of Lab
Investigation 5.3; and
A student guided inquiry lab on selecting another abiotic or biotic factor that
could affect the rate of enzyme reaction and conducting a student-designed
exploration in Part 2 of Lab Investigation 5.3.

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PREPARATION
Materials and Equipment are listed with each lab separately.
Timing and Length of Lab Activities

Lab Investigations 5.1 on Enzyme specificity, 5.2 on Rates of Reaction, and 5.3
on the effect of pH on enzyme activities are all using the same solutions prepared
in 5.1. Lab Investigations 5.1 and 5.3 could be done in one lab period. Rates of
reaction will take longer since measurements need to be taken every five minutes
for a total of 20 minutes. Student inquiry could take two to three lab periods,
depending on ExD (Experimental Design) development, conducting and collecting
data, graphing data, and discussing.

Learning objectives aligned to standards & science

Practices (SP)
To understand the relationship between enzyme structure and function (4B1
& SP 5.1).
To make some generalizations about enzymes by studying just one enzyme in
particular 4A1 & SP 6.1, 6.4).
To determine which factors can change the rate of an enzyme reaction and
which factors that affect enzyme activity could be biologically important (2D1
& SP 4.2, 7.2).

General Safety Precautions

Be sure to wear safety goggles when working with chemicals (pH buffers).
Since the concentration of the reactive materials in the lab investigations are
environmentally friendly, they can be rinsed down a standard laboratory drain.
Avoid rubbing your eyes or mouth when working with chemicals, even the lowest
concentrations can cause burning sensations to the eyes, lips, and mouth.

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Pre-lab Questions
Hydrogen peroxide, H2O2, is a toxic chemical that builds up in our bodies from
the metabolism of fats. Fortunately, our livers have a large quantity of the catalase
enzyme that breaks up the H2O2 produced from the metabolism of fatty acids in our
livers into harmless products of water and oxygen:
2H2O2 + Catalase enzyme 2H2O + O2 (g)

Other enzymes involved in the metabolism of certain amino acids and fatty
acids (D-amino acid oxidase and acyl-CoA oxidase) make significant amounts of
hydrogen peroxide. Since hydrogen peroxide can be damaging to normal tissue,
these enzymes are kept inside specialized organelles inside cells called peroxisomes.
The peroxisomes also contain large amounts of catalase enzyme to break down the
hydrogen peroxide before it can escape.
Catalase breaks down the hydrogen peroxide naturally produced by the body.
Studies have found that reduction in catalase production is correlated with gray hair,
suggesting that high hydrogen peroxide levels are behind gray hair. H2O2 can also
be damaging to cells, proteins and even DNA if it is not broken down efficiently in
our bodies.
If a cup of H2O2 is left standing in the open air for several days, eventually it
will spontaneously start to break up and oxygen gas will be released. However, if
we sprinkle a little dry yeast into a cup of H 2O2, rapid bubbling is observed. The
catalase enzyme in yeast is acting as a catalyst and lowering the activation energy
needed to drive this reaction, releasing oxygen gas. Another quality of a catalyst is
that it does not get used up in the reaction and recycles. The catalase enzyme can
decompose over a million H 2O2 molecules in less than a second.

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 1. The catalase enzyme is found in many plants and animals. What

do you think would happen to the protein enzyme catalase if the
yeast was boiled before adding to the H2O2?

2. What other environmental effects do you think could denature

the protein enzyme and cause it to be useless?

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LAB Investigation 5.1:

Enzyme Specificity
The monosaccharide glucose (simple sugar) is oxidized and decomposed
during cellular respiration for energy. As bonds are broken, 36-38 ATP molecules
are produced for each molecule of glucose oxidized (6-carbon simple sugar).
Enzymes are needed to decompose polysaccharides (starches, cellulose, glycogen),
disaccharides (2-sugar-12carbon compounds like lactose, sucrose, maltose), and
monosaccharides (a simple 6-carbon sugar like glucose, galactose, fructose) to
supply the bodys energy needs.
The most common disaccharides are sucrose (common table sugar) which is
composed of two simple sugars: glucose and fructose; and lactose (milk sugar) which
is composed of two simple sugars: glucose and galactose. In this investigation, the
enzyme lactase will be used in an attempt to break down the disaccharides sucrose
and lactose.

Materials:
Digital scale
Sucrose (table sugar), 1 gram*
Distilled water 250 mL*
Lactase tablet
Knife for cutting lactase tablet in half*
Spoons (for crushing the lactase tablet) (2)*
Whole milk, 1 mL*
24-well reaction plate
Pipets (8)
Glucose (dextrose), 2 grams
Glucose test strips (5)
Beaker plastic, 150 mL, 30 mL
Graduated cylinders, 10 mL
Test tubes, plastic with caps (4)
Test tube rack
Stopwatch
*items not included
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Preparations:
20% glucose (sometimes called dextrose a monosaccharide) solution: Add
2.0 grams of glucose to 10 mL distilled water in a plastic test tube, cap the test
tube and mix until it dissolves completely.
5% sucrose (table sugar a disaccharide) solution: Add 1.0 gram of sucrose to
20 mL of distilled water in a 30-mL plastic beaker, mix with a pipet until the
sucrose dissolves completely; transfer to a 12-mL test tube.
Lactase (milk sugar enzyme) solution crush half a lactase tablet between two
spoons over a 150-mL plastic beaker. Add 100 mL of distilled water. Mix
with a pipet until dissolved. Transfer 10 mL to a plastic test tube and cap to
save this for Lab 5.2 and Lab 5.3.

Procedures:
1. Fill in your predictions for qualitative results (using + or signs)
in data Table 5.1a.
2. Label and set up the following wells in a 24-well tray:
Table 5.1a

Well Plate
Label
A
B
C
D
E

Contents of each well:

3 drops sucrose solution +
3 drops distilled water
3 drops milk +
3 drops distilled water
3 drops sucrose solution +
3 drops lactase solution
3 drops milk +
3 drops lactase solution
3 drops glucose solution +
3 drops distilled water

Predictions for presence of

Glucose (+ or -)

Test Results Concentration

of Glucose(mg/dL)

Make sure you use a clean pipet for each solution including the
water and keep the pipet with the solution for later use.

3. Obtain a glucose test strip do not touch the test pad with your
fingers. Completely immerse a glucose test strip into well A
for 1-2 seconds, run the test strip along the edge of the well to
remove excess solution. (If the strip is NOT blotted, the glucose
concentration will not be accurate it may be too concentrated).

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 4. Follow the directions on the glucose test strip package for time
to wait before reading the test strip. Different manufacturers test
strips vary. The glucose test strips have specific waiting period.
Do not wait any longer than the prescribed time or your color
reading will not be accurate.
5. Follow the color chart on your glucose test strip packet. Record
the glucose concentration (based on the color chart, in mg/dL)
for well A in your data table.
6. Repeat Steps 2-4 for wells B, C, D, and E; using one new test
strip for each well.

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Analysis:
1. What was the enzyme in this reaction? The substrate? The
products? Explain why testing for glucose is used to determine
the activity of the enzyme lactase.

2. What determines a persons ability to digest lactose?

3. Explain the experimental conditions for the five different wells

and the results. Which wells showed positive for glucose? Do
they match your predictions?

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Lab Investigation 5.2: Enzyme Rate of

Reaction Assay and Baseline
To determine the rate of an enzymatic reaction, a change in the amount of
at least one specific substrate or product is measured over time. In this lab, we
will be monitoring the change in concentration of product (glucose) from the
decomposition of lactose to the simple sugars glucose and galactose. Glucose can
be measured and quantified by color and concentration as indicated by the glucose
test strip. A control will also be run with milk and water (no enzyme) as a baseline.
If there is any concentration of glucose in the control, it must be subtracted from
the experimental results.

Materials:
Glucose test strips (10)
Digital scale
Distilled water*
Whole milk, 1 mL*
24-well reaction plate
Stopwatch
Prepared Solutions from Lab Investigation 5.1
20% glucose
5% sucrose

Lactase Enzyme Solution

*item not included

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Procedures
1. Set up well A with 3 drops of Milk and 3 drops of distilled water,
this well will be the control well. Set up well B with 3 drops of
milk and 3 drops of lactase enzyme solution.
2. Immediately dip a test strip into Well A for 1-2 seconds and
record the concentration for Time 0. (Follow same procedures
for running the test strip along the edge of the well to remove
any excess. Read color chart at time indicated on the packaging
of the test strips. Record glucose concentration in your data
table. Follow the same procedure for Well B.
3. After 5 minutes, dip a new glucose test strip into Well A. Record
the glucose concentration. Repeat every 5 minutes. Take your
last glucose strip concentration at 20 minutes. Repeat for Well B.

4. Graph your results (time on the x-axis, glucose concentration on

the y-axis).

5. Calculate the rate based on the slope of a linear line during the
early period (Y/X). The slope of the graph during the early
period is called the initial rate of the reaction. As substrate
or enzyme is used up, the graph may plateau. The rate of the
reaction is the slope of the linear portion of the curve. The rate
will be expressed as (0.055)mg/dL glucose / sec = mmoles/L of
product (glucose) / sec.
Table 5.2a
Time
(min.)
0
5
10
15
20

Table: Presence of Glucose Indicating Lactase Activity

Rate of Reaction
Well A
Well B
(slope calculations)

6. IF there was any glucose concentration detected in Well A

during the 20 minutes, then you must use this as a baseline and
subtract it from the glucose concentrations found in Well B.

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Analysis:
1. Color change was measured at different times. What time
should be used for the pH assay in Lab Investigation 5.3? Why?

2. Describe the graph line shape? Was it possible to extrapolate a

straight line to determine the slope for the rate of reaction?

3. What was the result of the control? Will the experimental data
need to be adjusted?

4. Does the rate of reaction change or is it consistently the same for

every 5 minute interval?

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LAB Investigation 5.3

Part 1 - Determining the Effect of pH on

Enzymatic Activity
Changes in pH can alter the ionization of parts of the amino acids. This would
result in hydrogen bonding disruptions and the overall protein molecules 3D shape
and folding would change. This could alter the active sites and bonding ability
of the enzyme to the substrate. The enzyme could be nonfunctional if the pH is
drastic enough to denature it. In this investigation, you will explore the effects of
various pH values on the functioning of the lactase enzyme.

Preparations:
pH 4.0, 7.0, and 10.0 buffers: each tube contains approximately 0.1 gram of
powdered buffer. You need to add 10 mL of distilled water to make the buffer
solution. Gently rotate the tube to mix. The liquid buffer has a shelf life of
4 weeks if stored in air tight container; the powder has a shelf life of several
years. Using universal indicator strips, test each pH solution and record the
actual pH.
Materials:
pH buffer powder in capped test tubes: pH 4, 7, 10
Glucose test strips (6)
Whole milk*
Lactase solution (In capped test tube that you prepared in Lab 5.1)
24-well reaction plate
Stopwatch
Pipets (5)
Test Tubes, plastic with caps (5)
Universal Indicator strips (3)
*item not included

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Procedures:
Table 5.3a
Wells

A
B
C

1. Set up the wells according to the data chart:

Contents of Wells
2 drops Buffer pH 4
+ 2 drops Lactase + 2
drops Milk
2 drops Buffer pH 7
+ 2 drops Lactase + 2
drops Milk
2 drops Buffer pH 10
+ 2 drops Lactase + 2
drops Milk

pH

Concentration of
Concentration of
Glucose at 0 min.
Glucose at 10 min. mg/
mg/dL(0.055) = mmol/L dL(0.055) = mmol/L

4
7
10

2. Record initial glucose concentrations at Time 0. Wait 10 minutes

for the reactions to occur and dip a new glucose test strip.

3. Follow the same procedures for completely immersing the test

pad in the well for 1 second. Run the test strip along the edge of
the well to remove excess liquid. Wait test strip instructed time
for the color to develop. Then compare the test strip to the color
chart for concentration. Record the glucose concentration in
Table 5.3a at Time 0 and at Time 10 min..

4. Test the remaining wells, using a new test strip for each well and
record the glucose concentrations at Time 0 and Time 10 min..
5. Graph the data placing glucose on the y-axis and the pH values
on the x-axis.

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Data Analysis:
1. What conclusions can be draws from the results?

2. What was the effect of pH on the enzyme lactase? Is this true

for all enzymes?

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Part 2 - Student Guided Inquiry

Student Guided Inquiry of the Environmental Effects on Enzyme Activity
What are three or four factors that vary in the environment in which organisms
live? Which of those factors do you think could affect enzyme activity?
Following are suggestions for a student guided inquiry:
Effect of Various Temperatures on Enzyme Rates of Reaction
Effect of Various Enzyme and Substrate Concentrations on Rates of Reaction:
Which has a greater effect on the rate of reaction changing the concentration
of enzyme or changing the concentration of substrate?
Effect of Enzymes from Different Organisms (lactase from fungi vs. lactase
from humans)
How are organisms survival linked to enzymes?

Effect of Various Soil Salinities with different plants/

seeds
Effect of Various Soil pH with different plants/seeds
Effect of light or no light in germination of seeds

Design and conduct an experiment to investigate one of the biotic or abiotic

environmental factors that influence the rate of enzymatic reactions.
Step 1:

Chose your driving question to investigate.

Step 2:

Fill in the ExD (Experimental Design) form on the next page to

plan your experiment.
Step 3:

Design data tables and collect data.

Step 4:

Analyze the data.

Step 5:

Summarize your conclusions.

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Experimental Design (ExD) Form

Complete this ExD pre-lab planning form before beginning your lab
1.

Independent variable: (What is the cause agent? What are you changing?)

2.

Dependent variable: (What is being measured?)

3.

Lab set-up:
Experimental
Groups
Number of Trials

4.

Control: (What is the experimental group being compared to?)

5.
Hypothesis: (Use an if [Independent Variable], then [Dependent Variable] format.
State the cause and effect relationship between the I.V. and the D.V. It must be testable.)

6.

Lab title: (The effect of ____[I.V.] ____on ____[D.V.]____.)

7.

Experimental constants: (Name at least six variables NOT altered during the experiment.)

8.

Sketch of experimental set up with labels:

9.

Detailed procedure:

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