BSC 2010L – Principles of Biology 1 Lab
Lab Topic 4
Enzymes
Lab Topic 4: Enzymes
• Laboratory Objectives
– After completing this lab topic, you should be able to:
• 1. Define enzyme and describe the activity of enzymes in
•
•
•
•
•
cells.
2. Differentiate competitive and noncompetitive inhibition.
3. Discuss the effects of varying environmental conditions
such as pH and temperature on the rate of enzyme activity.
4. Discuss the effects of varying enzyme and substrate
concentrations on the rate of enzyme activity.
5. Discuss the scientific process, propose questions and
hypotheses, and make predictions based on hypotheses and
experimental design.
6. Practice scientific thinking and communication by
constructing and interpreting graphs of enzyme activity.
Activation energy
Enzyme added
Reactant
Reactant
Products
Products
Introduction
• Living cells perform a multitude of chemical
reactions very rapidly because of enzymes
• Enzymes are biological catalysts that speed up a
chemical reaction without being used up or
altered in the reaction
– Enzymes are proteins and show high specificity for
particular substrates
• The substrate is the material with which the
catalyst reacts and is modified during the reaction
to form a new product
• The active site of an enzyme will bind to the
substrate, forming the enzyme-substrate complex
• Enzymes lower the energy activation barrier and
thus accelerate the rates of reactions
• Enzyme activity is influenced by many factors:
pH, temperature, activators, inhibitors
(competitive and non-competitive), and cofactors
(coenzymes, metal ions)
Enzymes and activation energy
Activation
energy barrier
Reactant
Energy level
Energy level
Enzyme
Reactant
Products
(a) Without enzyme
Activation
energy barrier
reduced by
enzyme
Products
(b) With enzyme
How an enzyme works
Substrate (sucrose)
Active site
Sucrase can accept a
molecule of its substrate.
Substrate binds
to the enzyme.
Enzyme
(sucrase)
Fructose
H2O
Glucose
The products
are released.
The enzyme
catalyzes the
chemical reaction.
Induced Fit Between an Enzyme and its Substrate
Substrate
Active site
Enzyme
(a)
Enzyme-substrate
complex
(b)
1 Substrates enter active site; enzyme
changes shape such that its active site
enfolds the substrates (induced fit).
2 Substrates held in
active site by weak
interactions, such as
hydrogen bonds and
ionic bonds.
Substrates
Enzyme-substrate
complex
6 Active
site is
available
for two new
substrate
molecules.
Enzyme
5 Products are
released.
4 Substrates are
converted to
products.
Products
3 Active site can lower EA
and speed up a reaction.
A
B
C
D
Transition state
A
B
C
D
EA
Reactants
A
B
∆G < O
C
D
Products
Progress of the reaction
Course of
reaction
without
enzyme
EA
without
enzyme
EA with
enzyme
is lower
Reactants
Course of
reaction
with enzyme
∆G is unaffected
by enzyme
Products
Progress of the reaction
Exercise 4.1 – Experimental Method and the
Action of Catechol Oxidase
• Materials
– See page 84
• Introduction
– This exercise will investigate the result of catechol oxidase
activity
• Question
– Formulate a question about catechol oxidase and potato extract
• Hypothesis
– Construct a hypothesis for the presence of catechol oxidase in
potato extract
• Prediction
– Predict the result of the experiment based on your hypothesis
• Procedure
– See page 86
• Results
– Complete Table 4.2 on page 87
• Discussion
– Explain your results in terms of your hypothesis on page 87
The oxidation of catechol
Exercise 4.2 – Inhibiting the Action of
Catechol Oxidase
• Materials
– See page 87
• Introduction
– This exercise will investigate the inhibition of enzyme activity by
specific chemicals called inhibitors
– The specific inhibitor to be investigated is phenylthiourea (PTU)
• Question
– Pose a question about the activity of PTU
• Hypothesis
– Hypothesize about the nature of inhibition by PTU
• Prediction
– Predict the results of the experiment based on your hypothesis
• Procedure
– Follow the procedures on pages 88-89
• Results
– Record your results in Table 4.4
• Discussion
– Answer questions 1-2 on page 89
(a) Enzyme and substrate
binding normally
Substrate
Active site
(b) Enzyme inhibition by
a substrate imposter
Enzyme
Substrate
Inhibitor
Active site
Enzyme
(c) Enzyme inhibition by
a molecule that causes
the active site to change
shape
Active site
Substrate
Inhibitor
Enzyme
Exercise 4.3 – Influence of Concentration, pH,
and Temperature on the Activity of Amylase
• Introduction
• Experiment A. The Influence of Enzyme Concentration on the
Rate of Starch Digestion
– Materials
• See page 90
– Introduction
• In this experiment we will vary the concentration of the enzyme amylase
to determine what effect the variation will have on the rate of the
reaction.
– Question
• Pose a question about enzyme concentration and reaction rate.
– Hypothesis
• Hypothesize about the effect of changing enzyme concentration on the
rate of reaction.
– Prediction
• Predict the results of the experiment based on your hypothesis
– Procedure (pages 91-92)
– Results
• Complete Table 4.5 and Figure 4.5 on pages 92-93
– Discussion
• Answer questions 1-2 on page 94
Table 4.5
Time of Starch Disappearance in Different Concentrations of the Enzyme Amylase
Tube
% Amylase
Time of Starch Disappearance (in seconds)
1
0.50
60
2
0.25
70
3
0.125
90
4
0.063
180
5
0.031
360
Exercise 4.3 – Influence of Concentration, pH,
and Temperature on the Activity of Amylase
• Experiment B. The Effect of pH on Amylase Activity
– Materials
• See page 94
– Introduction
• In this experiment we will determine the optimum pH for the
activity of amylase
– Question
• Pose a question about pH and reaction rate
– Hypothesis
• Hypothesize about the rate of activity of amylase at various
levels of pH
– Prediction
• Predict the results of the experiment based on your hypothesis
– Procedure (page 95)
– Results
• Complete Table 4.6 and Figure 4.6 on pages 96-97
– Discussion
• Answer the question on page 97
Table 4.6
Time of Starch Disappearance in Different pH Environments for the Enzyme Amylase
Tube
pH
Time of Starch Disappearance (in minutes)
1
4
10
2
5
8
3
6
3
4
7
4
5
8
7
6
9
10
Exercise 4.3 – Influence of Concentration, pH,
and Temperature on the Activity of Amylase
• Experiment C. The Effect of Temperature on Amylase
Activity
– Materials (page 97)
– Introduction
• This experiment will study the effects of temperature on
amylase activity
– Question
• Pose a question about temperature and reaction rate
– Hypothesis
• Hypothesize about the rate of activity of amylase at various
temperatures
– Prediction
• Predict the results of the experiment based on your hypothesis
– Procedure (see pages 98-99)
– Results
• Complete Table 4.7 and Figure 4.7 on pages 99-100
– Discussion
• Answer questions 1-2 on pages 100-101
Table 4.7
Time of Starch Disappearance in Different Temperatures for the Enzyme Amylase
Tube
Temp. °C
Time of Starch Disappearance (in minutes)
1
80
2
37
3
3
22
7
4
4
8
Rate of reaction
Optimal temperature for
typical human enzyme
Optimal temperature for
enzyme of thermophilic
(heat-tolerant)
bacteria
40
60
80
Temperature (ºC)
(a) Optimal temperature for two enzymes
0
20
Optimal pH for pepsin
(stomach enzyme)
100
Optimal pH
for trypsin
Rate of reaction
(intestinal
enzyme)
4
5
pH
(b) Optimal pH for two enzymes
0
1
2
3
6
7
8
9
10
Lab Topic 4: Enzymes
• Reviewing Your Knowledge
– Answer questions 1-2 on page 101 in your lab
manual
• Applying Your Knowledge
– Answer questions 1-5 on pages 101-102 in
your lab manual