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EXERCISE 4

ENZYMES

Functional and Structural Analysis

OBJECTIVES

Upon the completion of this exercise, the student should be able to:

1. design experiment(s) with appropriate controls to test your hypothesis.
2. make a standard curve for estimation of maltose concentration.
3. set up enzyme-catalyzed (amylase) hydrolysis of starch into maltose.
4. explain the relationship between amount of maltose produced and the activity of amylase.
5. obtain amino acid sequences of amylase from the NCBI online database.
6. use bioinformatics tools to analyze primary structures, compare amino acid sequences and

generate a 3D structure of each enzyme.

The following videos have been prepared to help you better understand the structure of this lab. It is very
important to watch these videos and write notes before starting the lab activity.

1. Introduction to enzymes’ role in biochemical reactions: a review of proteins/enzymes, the
amylase enzyme, and the reaction it catalyzes.
https://www.youtube.com/watch?v=V6wHXtO9klA

2. Maltose standard curve: constructing and using this graph will allow you to determine amylase
activity. https://youtu.be/KNMz0pSgYbk

3. Testing your hypothesis on amylase activity: review of the scientific method, the experimental
design, and data analysis to determine the effect of an environmental factor on amylase activity.

Chemical reactions that take place in the cell do not occur randomly, they are controlled by biological
catalysts, called enzymes. A catalyst is a substance that speeds up the rate of a chemical reaction without
being used up by the reaction. Most enzymes are proteins whose primary structure is dictated by genes.
Enzymes do not become active until the polypeptide chains are folded into a unique three-dimensional
(3D) shape. The 3D structures are held together by various non-covalent interactions such as hydrogen
bonds, ionic bonds, disulfide bridges, hydrophobic and Van der Waals

Thousands of product molecules may be formed in one second by an individual enzyme.
Reactants in an enzymatic reaction are called substrates and substrate(s) bind to the enzyme at a
specific site, called the active site. Active site of the enzyme is like a pocket or cleft in the
protein that is shaped in a way that substrates can fit in. Since only properly shaped substrates
can fit into the active site, specific enzymes bind specific substrates, similar to a key that is
shaped to fit a specific lock. Some enzymes may have metal ions (e.g., Ca2+, Cu2+, Fe2+, Mn2+)
as part of their active site and are called cofactors. The substrate(s) is converted into a product at
the active site. Substrates bind to enzyme molecules using a combination of weak, noncovalent

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chemical bonds, forming an enzyme-
substrate (ES) complex that exists for a
fraction of a second. During this time,
subtle changes in the shape of the active
site, called induced fit, stress and orient
covalent bonds of the substrates in a way
that facilitates the formation of product(s)
(Figure 1). The newly-formed product
then leaves the active site, whereas the
enzyme remains unchanged and can bind to
additional substrate molecules, if they are
available.

Enzymes speed up the rate of a reaction by
lowering the initial amount of energy required,
called the activation energy (EA). Activation
energy is required by all chemical reactions to
break certain bonds (free up electrons) in the
reactant(s) so that new bonds can form, resulting in
product formation (Figure 2). Enzymes use energy
from the surrounding environment to increase
molecular movement of substrates. This reduces
the activation energy barrier.

The activity of enzymes, like that of all proteins, is
affected by environmental conditions. Factors like
temperature, pH, and salt concentration interfere
with the non-covalent forces that give enzymes their
3D shape and enzymes then start to unfold, or denature. Alpha helices and beta sheets, for example, are
disrupted in denaturing conditions, and the peptide chain takes on a random shape. Denaturation does not
break peptide bonds, so the enzyme’s primary structure is unaffected. However, because the enzyme has
changed shape, the active site is also altered and will not complement and bind the substrate as well. The
reaction rate therefore decreases accordingly. Enzymes can structurally be similar, and catalyze the same
reaction, yet they may have important differences leading to different optimal temperatures.

Organisms regulate the rates of their reactions by regulating the activity of their enzymes. Most enzymes are
adapted to function in specific conditions. (almost a repeat of the last sentence in the previous paragraph).

Figure 2: Energy diagram of a reaction in the
presence and absence of enzyme
Originally uploaded by Jerry Crimson Mann, vectorized by
Tutmosis, corrected by Fvasconcellos / CC BY-SA
(http://creativecommons.org/licenses/by-sa/3.0/)

Figure 1: Induced fit mechanism of enzyme action

(Created by TimVickers, vectorized by Fvasconcellos / Public domain)

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Factors that affect the rate of enzyme activity:

1. Temperature affects enzyme activity in two ways. As the temperature rises, molecular motion
(kinetic energy) increases and the rate of random collision between enzyme and substrate
molecules increases, forming more products. After a certain point, increasing the temperature
strains the non-covalent bonds, altering the shape of the active site, and the overall shape of the
enzyme. This decreases the rate of product formation. The temperature at which enzyme activity
is the highest is called the optimum temperature. At high temperature, an enzyme will most
likely unfold and denature.

2. Changes in pH (H+ concentration) and salt concentration primarily affect the stability of

secondary and tertiary structures maintained by hydrogen bonds and disrupt salt bridges held by
ionic bonds. As a result, enzymes denature at extreme pH and high salt concentrations. In
addition, substrates and/or enzyme active site groups may ionize, which further affects enzyme-
substrate binding.

3. Substrate and enzyme concentration also affect the rate of enzyme reaction. Increasing the

concentration of substrate and/or enzyme increases the rate of reaction up to a certain point. As
the reaction continues and the substrate molecules are used up, the rate of reaction will decrease
regardless of any changes in enzyme concentration. By controlling enzyme and substrate
concentration, organisms can regulate their metabolism.

ENZYME ACTIVITY IS MEASURED BY MONITORING CHANGES IN SUBSTRATE AND/OR
PRODUCT CONCENTRATIONS.

To learn about the function and structure of enzymes, we will be using the enzyme amylase as a model in this
lab. Alpha amylase, is an enzyme that catalyzes the breakdown (hydrolysis) of α-1,4 glycosidic linkages of
starch (a polymer of glucose), into maltose (a reducing disaccharide made of two glucose molecules).
Because starch is one the most abundant carbohydrate polymers on earth, it serves as a major source of
energy not only for us but for many other animals, higher plants and microorganisms as well. In order to
harvest the energy from starch, the enzyme amylase, present in the saliva and pancreatic secretions of
humans and other mammals, begins the chemical process of breaking (or hydrolyzing) it down into smaller
sugars. Amylase is present in all 3 domains of life – Bacteria, Archaea and Eukarya (plants, animals, and
fungi) – with the same catalytic function.

Starch + water Maltose
amylase

Alpha amylases from three different sources, (i) bacteria (Geobacillus stearothermophilus), (ii) fungi
(Aspergillus oryzae), and (iii) humans (Homo sapiens), will be used in this lab.

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Concentration of maltose can be measured using a colorimetric assay: that is, combining maltose with a
certain reagent that causes a color change. The reagent in this assay is DNS (3,5-dinitrosalicylic acid, also
called DNSA) which is yellow in color. First introduced to detect reducing substances in urine, the DNS
assay is commonly used to quantitate carbohydrate levels in blood as well as detecting alpha amylase
activity.

In an alkaline solution, reducing sugars form aldehyde or ketone groups, which can then reduce different
reagents (e.g., Benedicts reagent, dinitrosalicylic acid or DNS). Unlike Benedicts test for detecting the
presence of reducing sugars, reaction between DNS and a reducing sugar results in a soluble and colored
product. Maltose participates in an oxidation-reduction reaction with DNS due to the presence of a carbonyl
group (C=O). DNS is reduced to 3-amino, 5-nitrosalicylic acid (ANSA – will be referred to simply as
reduced DNS) and maltose is oxidized to maltonic acid (Figure 3).

This reaction causes a change in color from yellow to orange/red when DNS is reduced (Figure 3). The
change in color results in a change in absorption of light, and absorbance is measured in a
spectrophotometer at a wavelength of 540 nm. The intensity of color from the reaction, and the
absorbance of light, is proportional to the concentration of maltose and is used to estimate the
concentration of maltose in any given solution.

The concentration of maltose produced by hydrolysis of starch is directly dependent on amylase
activity. Therefore, absorbance data can be used to determine the optimal conditions for amylase
activity, i.e., conditions at which this enzyme has the highest activity.

redox reaction

Figure 3: Reduction of DNS by maltose produces reduced DNS and maltonic acid.

Maltose Maltonic acid

NEUROtiker /
P bli d i

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PRE-LAB QUESTIONS:
SUBMIT ONLINE OR HAND IN AT THE BEGINNING OF YOUR LAB

1. Define Enzymes. What type of macromolecules are enzymes?

2. What are 2 advantages of having enzyme-catalyzed chemical reactions in living
cells?

3. What is a substrate? Where on an enzyme does the substrate specifically bind to
during a chemical reaction?

4. When an enzymatic reaction is in progress, do you expect to see an increase, decrease or
no change in each of following:

(i) substrate

(ii) product

(iii) enzyme

5. How can you measure enzyme activity?

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6. What is the relationship between enzyme activity and 3D shape of an enzyme? What type of
environment changes affect the function of an enzyme?

7. Define optimal conditions for enzyme activity. How can you determine (i) optimal temperature
and (ii) optimal pH of an enzyme?

8. What enzyme will you be studying in lab today? Where can you find this enzyme?

9. Write the reaction catalyzed by amylase. Do you expect this enzyme to hydrolyze cellulose (a
polymer of glucose)? Explain.

10. Production of maltose, by ___________________ condensation, dehydration,
hydrolysis) of starch, in the presence of amylase, can be detected by
___________ assay.

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PART I: INTRODUCTION TO THE ASSAY FOR HYDROLYSIS OF STARCH BY
AMYLASE

In this part of the exercise, you will be introduced to the hydrolysis of starch, a reaction catalyzed by
amylase and the DNS assay used to measure amylase activity. You set up hydrolysis of 5% starch in
vitro, at room temperature (25°C), using fungal amylase. Remember this may not be the optimal
temperature for this enzyme. ACTIVITY OF AMYLASE IS DIRECTLY RELATED TO THE
AMOUNT OF MALTOSE PRODUCED.

EXPERIMENTAL SETUP:

1. Two tubes are prepared according to the table below. For a video on how to use micropipettes,
click here:

Tube Water (μl) 5% Starch
(μl)

Fungal Amylase
(μl)

Total Volume (μl)

A 450 450 100

B 550 450 0

2. Both tubes are incubated for 10 min at 25°C (on bench).

3. 1000 μl (1 ml) DNS is added and the tubes are placed in boiling water for 5 min.

4. 8 ml of dH2O (deionized water) is added to both samples to dilute them, using a serological
pipet. The absorbance of light at 540 nm is read for the two solutions using a
spectrophotometer. (Do we need this? It will be in Lab 1)Here is the link to a video on how to
use a spectrophotometer like the one we have in our lab:

Tube B is used as a “blank”, to calibrate the machine.

What treatment is tube B in the experiment? _________________________

What treatment is tube A in the experiment? _________________________

5. The absorbance of tube A you read in the spectrophotometer is 0.358.

6. After completing Part II, the maltose standard curve, determine the maltose concentration in your
tube ________________.

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PART II: STANDARD CURVE FOR ESTIMATION OF MALTOSE

The objective of this part of the lab activity is to collect data and plot a standard curve. The procedure
for this activity is described below:

DNS is added to known amounts of maltose, and the absorbance of the reduced DNS is measured at
540nm, using a spectrophotometer. A reference graph, called a standard curve, is made from this data.
A standard curve is a graph which shows a relationship between two quantities; in this activity, the
relationship between known concentrations of maltose and their absorbance. You may want to review
the “line of best fit” section in the graphing skills part of Exercise 1. In part III of this lab exercise, you
will use the standard curve to estimate the amount of maltose produced during hydrolysis of
starch, the reaction catalyzed by amylase. The data on the amount of maltose produced will then
be used to calculate amylase activity.

To prepare the standard curve, the concentration of maltose (mg/ml) in each tube has to be determined
first. Please watch this video https://youtu.be/KNMz0pSgYbk to understand the steps involved in
calculations, and to construct the standard curve.

1. Complete the table below and show a sample of your calculations. You will be using the

formula CiVi = CfVf (equivalent to C1V1 = C2V2).

The initial maltose concentration (Ci) used is 2.5 mg/ml. After adding 1 ml DNS to 1 ml of maltose,
all samples are diluted with 8 ml of water (do you want to mention about the redox reaction
occurring at a higher temperature?), and their absorbance is determined in a spectrophotometer and
indicated in the table. The final volume of maltose (Vf) is, therefore, 10 ml.

Table 1: Final Concentration of maltose

Volume of
initial
maltose
solution Vi
(μl)

Volume of
initial
maltose
solution Vi
(ml )

Volume of
water added
to get a total
of 1 ml

Final Maltose
Concentration
(mg/ml)

Absorbance
at 540nm

0

0.000

200

0.228

400

0.487

600

0.751

800

1.031

1000

1.275

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2. Graph 1: graph the Absorbance vs. Concentration of maltose from Table 1. There are two
ways to do it, a) manually, on graph paper, OR b) in Microsoft Office Excel, depending on
what your instructor has asked you to do. Remember to use TAILS and insert your graph below. If
you are not sure how to do that, watch the video on maltose standard curve for help.

Choose one of these two ways to present your graph:

a. Construct your graph manually, on graph paper. Draw a line of best fit through your points,
making sure that line passes closer to as many data points as possible. Embed a picture
below. This graph will be used in Part III, to determine the amount of maltose produced in
your experimental tubes.

OR

b. Construct your graph on excel, according to the instructions on the video provided on the
maltose standard curve. Draw a ‘line of best fit’ through your data points by clicking on a
point and selecting “add trendline”. In the menu that opens, select “display equation of line,
to get the equation of the line to appear on your graph. Embed your excel graph below. You
will use this equation of the line in Part III, to determine maltose produced in your
experimental tubes.

Part III: Test your hypothesis

Before doing this part, watch this video https://youtu.be/_BacifzIP64 on testing your hypothesis on the
effect of a preassigned variable (temperature, for example) on amylase activity. You will be assigned an
independent variable and amylase enzyme isolated from one of the three organisms described in the
background information and the introduction video.

You will write a question, a hypothesis and a prediction based on the source of amylase (bacterial,
fungal or human) and the independent variable you were assigned. You will also define the
dependent variable. To write a good hypothesis, research the literature on the organism/enzyme
you are studying, and base your hypothesis on the information you find. Make sure to cite your
sources (APA style) both in the text and at the end of your report.

Remember:

• Question: Asking a question is an important part of the scientific process. The more specific

questions you ask, the easier it will be for you to design experiments to test your hypothesis.

• Hypothesis: Tentative answer to the question you asked in the previous step. Write a simple but

specific statement that is testable and falsifiable. You should research the literature on the
organism/enzyme you are studying, and base your hypothesis on the information you find.
Make sure to cite your sources (APA style) both in the text and at the end of your report.

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• Prediction: The prediction describes what will happen if a hypothesis is correct. Generate a
reasonable prediction by completing this statement: If _____ then ____.

Determine which amylase and which independent variable you are supposed to study, based on
your last name, according to the table below:

LAST INITIAL AMYLASE INDEPENDENT VARIABLE

A-D Geobacillus stearothermophilus

(bacterial)

Temperature

E-H Aspergillus oryzae (fungal)

pH

I-L Homo sapiens (human) pH

M-P Geobacillus stearothermophilus

(bacterial)
pH

Q-T Aspergillus oryzae (fungal) Temperature

V-Z Homo sapiens Temperature

3. Experimental Design: Table 2A shows how your tubes would be set up if you were studying the

effect of temperature on amylase activity. Table 2B shows how your tubes would be set up if you
were studying the effect of pH on amylase activity.

• Tube contents are mixed, and incubated in water baths at appropriate temperatures for 10
minutes.

• 1 ml DNS is added to all tubes and they are boiled for 5 minutes.

• 8 ml of water is added for diluting, and absorbance readings are collected in a
spectrophotometer as indicated in Table 3.

Table 2A: experimental setup for Temperature
Tube Water (μl) 5% Starch (μl) Amylase (μl) Temperature (0C)

1 450 450 100 0

2 450 450 100 25

3 450 450 100 37

4 450 450 100 45

5 450 450 100 65

6 450 450 100 85

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Table 2B: Experimental setup for pH
Tube Buffer (μl) 5% Starch (μl) Amylase (μl) pH

1 450 450 100 1

2 450 450 100 3

3 450 450 100 5

4 450 450 100 7

5 450 450 100 9

6 450 450 100 12

The tubes listed in the table are your experimental treatment. What will the negative control tubes
contain?

4. From the tables in the Appendix, choose the one that contains data for your amylase enzyme
and your independent variable, according to the determination you made based on your last name.
Insert the appropriate table in your report and delete the Appendix. Using either the manual graph,
or the excel graph of the maltose standard curve you constructed in Part II
(https://youtu.be/KNMz0pSgYbk):
a. Determine the maltose produced in your experiment and fill the data in Table 3. Show an

example calculation below the table.
b. Determine the amylase activity in each tube and fill in that data in Table 3. Show an

example calculation below the table.

5. Graph 2: graph the resulting Amylase Activity data vs. your Independent Variable (on Excel
or manually on graph paper) and insert your graph or a picture of it in your report.

6. Conclusion: restate your hypothesis and summarize your results based on your graph of the

Amylase Activity vs. your Independent variable. Explain whether your graph supports your
hypothesis, by using information from the graph. Comment on other findings from the graph.
Comment on what you think happened to the Amylase enzyme and its structure and function at
points where you observe low activity.

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Rubric Enzyme Report (40 points)

1. Introduction: (4 pts)

a. What are enzymes and what do they do?

b. What are the optimal conditions for an enzyme?

c. What factors affect enzyme activity and how?

d. How is the structure of an enzyme related to its function?

e. What is amylase? What reaction does it catalyze?

f. Research the amylase enzyme you used (organism) and the optimal conditions of temperature,
pH, or salt, it requires.

g. Explain why DNS is used and why it is important for determining enzyme activity. Cite and
reference your sources using APA style.

2. Experimental Design: (5 pts)

a. State your question, hypothesis (ensure that it is testable and falsifiable), and prediction.

b. Identify the independent variable.

c. Identify the dependent variable.

d. Identify the experimental group

e. Identify the control group.

3. Materials/Methods: (3 pts)

List all of the equipment and materials you will need for your experiment. BRIEFLY describe your
experimental procedure for setting up the maltose standard curve (Part II) and for testing your
hypothesis on amylase (Part III).

4. Data Analysis/Results:

a. List all the formulas you used for your calculations and a sample calculation in each case (3 pts)

b. Prepare the following tables:

• Table 1 with maltose standard curve data. (3 pts)

• Table 2 with your experimental setup. Explain how your control tubes will be set up. (2 pts)

• Table 3 with absorbance measurements of experimental tubes, calculated maltose
concentration, calculated amylase activity (mg/ml maltose/min) (4 pts)

c. Prepare the following graphs:

• Graph 1 of maltose standard curve: show line of best fit and equation of line if done on
excel. If done manually, insert an image of your graph indicating the line of best fit. (3 pts)

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• Graph 2 of enzyme activity versus independent variable (4 pts)

d. Summary of results: summarize your results based on your graphs (3 pts)

5. Conclusion/Discussion: (4 pts)
Based on your results, is your hypothesis supported? Restate your hypothesis and explain! Discuss
any other findings and explain them. Discuss errors, and reasons for data variability.

6. References: (2 pts)
List the sources you used at the end of your report, and include in-text citations in your report (APA
citation style)

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PART IV: BIOINFORMATICS – STRUCTURAL ANALYSIS OF ALPHA AMYLASES

Bioinformatics is the use of computer software and computational tools, for the analysis of protein and
nucleic acid sequence information (online databases), through the use of sophisticated, stand-alone and
widely available online software packages.

This part of the lab exercise
will introduce you to
elementary bioinformatics tools
for the analysis of three alpha
amylase enzymes that were
used in parts I-III. You will
retrieve the amino acid
sequences of all three amylases
from an online databank,
analyze those sequences and
view their three-dimensional
structures. Since online
databanks use the one-letter
amino acid code, please look at
the figure, on your right, in
order to familiarize yourself
with the one-letter system.

Ability to maintain functional
3D structure within a particular
range of temperature, pH,
salinity, is an intrinsic property
of proteins and is determined
by the primary structure.
Amino acid sequence
determines the way a protein
folds into unique 3D shape
(native conformation). Protein
homology means that the proteins are derived from a common ancestor gene and will have the same
number and types of secondary structure, oriented in the same way in three-dimensional space. All
amylases catalyze hydrolysis of starch, yet their amino acid sequences have significant differences
among different organisms.

Increased thermostability can be conferred by a greater number of ionic interactions, disulfide bridges,
proline residues, as well as numerous other structural strategies. We will also discuss the factors that
give rise to protein thermostability and interpret the data in an evolutionary context.

In many ways, biology has become a “big data” science. Since the human genome project was
completed, many more genomes from all domains of life have been sequenced. Furthermore, scientists
routinely sequence individual genes as well as their associated proteins. That wealth of genomic data,

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which includes RNA sequences and non-coding DNA sequences, is stored in a number of databases
housed by the National Center for Biotechnology Information or NCBI (http://www.ncbi.nlm.nih.gov/).
While the NCBI is known mostly for its searchable databases, another website known as ExPASy
provides scientists with computational tools to analyze genomic data. ExPASy bioinformatics portal
(http://www.expasy.org/) is maintained by the Swiss Institute of Bioinformatics (SIB). Another popular
bioinformatics portal is The European Molecular Biology Open Software Suite or EMBOSS
(http://emboss.sourceforge.net/) which is a product of the European Molecular Biology network.

In part III, you tested your hypotheses on the effect of different variables on alpha-amylase activity.
Now, let’s get some insight on the evolution of thermostability of alpha-amylase. We will use various
biocomputational tools to compare the primary, secondary and tertiary structures of human,
fungal and bacterial alpha-amylases. We will then discuss the factors that may contribute to
thermostability and the evolutionary mechanisms that may have led to thermostability of alpha-amylase.
Retrieval of the amino acid sequences of amylases: In order to analyze our proteins we must first
retrieve their amino acid sequences from the NCBI database. As in any archive or library where items
are given an accession number for easy retrieval, protein and DNA sequences in the NCBI database are
associated with an accession number as a unique identifier (indicated below, in Table 4). The table also
includes another unique identifier for the Protein Data Bank or PDB, one of the databases that will be
used in this exercise.

Table 4: NCBI ACCESSION NUMBERS AND PDB ID’S OF ALPHA AMYLASES

Source of alpha amylase NCBI accession number PDB ID

Homo sapiens (salivary) GI:157833830 1SMD

Aspergillus oryzae GI:541881321 3VX0

Geobacillus stearothermophilus GI:8569361 1QHP

Analysis of primary structures of amylases: One of the software tools from EMBOSS, named
PEPSTATS, computes statistics on the properties of individual proteins based on their primary
structures. This tool can provide information about the nature of the amino acids (with polar, nonpolar,
or charged side chains, etc).

Multiple sequence alignment of amylases: Sequence alignments can be used to detect homology. A
multiple sequence alignment is a computational method of aligning two or more monomeric sequences
of proteins or nucleic acids in order to (i) compare identities and similarities and (ii) determine
evolutionary relationship. It can also be used when a novel protein is discovered and information is
needed about its function. Homologous sequences usually have similar structure and function.
Significant sequence homology and structural similarity strongly implies common ancestry.
Functional similarity ‘supports’ common ancestry but is not sufficient to demonstrate it. Sequence
homology, expressed in percentage, is based on similarity or identity in the amino acid sequence.
Similarity means that the residues in a pair of sequences are chemically similar, and identity means that
the residues are exactly the same. Similar amino acids can replace one another over the course of an

http://www.expasy.org/

http://emboss.sourceforge.net/

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evolutionary period and still have the same function.

One of the most reliable and versatile bioinformatics tools is called CLUSTAL OMEGA, offered by the
European Bioinformatics Institute (EBI). CLUSTAL OMEGA is a general purpose multiple sequence
alignment program for DNA/proteins, for alignment of 3 or more sequences. It calculates the best match
for the selected sequences, and lines them up so that the identities, similarities and differences can be
seen. Evolutionary relationships can also be determined by viewing Cladograms or Phylograms.

Highlighting conserved sequences in the sequence alignment: In sequence alignment, certain amino
acid residues are more important to the function than others and thus are highly conserved throughout
the evolutionary history. The multiple sequence alignment is more useful when it can be viewed with an
editor such as BOXSHADE. This is an alignment visualization software that highlights identical amino
acids across sequences with black boxes, and similar amino acids with grey boxes.

The ability to compare amino acid sequences between different organisms allows us to see the
conserved regions and infer that those sequences may be involved in the function of proteins. The exact
roles of highly conserved active site amino acid residues in alpha amylases in the catalytic process is not
completely clear. Diversity in amino acid sequences of the 3 amylases reflect flexibility to change amino
acids to optimize enzymatic activity under the particular condition that each alpha amylase is required to
function.

Three-dimensional structures of amylases: A protein’s three-dimensional structure determines its
function. The 3D structures of a number of proteins have been determined by X-ray crystallography
and/or Nuclear Magnetic Resonance spectroscopy. As stated earlier, the NCBI stores the sequences of
millions of proteins and nucleic acids; there is also a repository for the three-dimensional structures of
many of those proteins, and it is known as the Protein Data Bank or PDB. As in the NCBI database,
each 3D structure is assigned an accession number so that it can be easily found in the databank. That
number is known as the PBD ID; the PDB IDs of human, fungal and bacterial alpha amylases can be
found in Table 4.

As a polypeptide starts to fold, secondary structures, such as alpha helices, beta sheets, turns, and loops,
start to form. Secondary structures form in parts of a polypeptide with the help of hydrogen bonds. Tertiary
structure is the total 3D conformation (shape) of an entire polypeptide chain including, alpha helices,
beta sheets and any other loops, turns or bends.

Organisms found at low temperatures have membrane proteins with a higher percentage of alpha helices
(provides flexibility) compared to beta sheets (provides rigidity). On the other hand, thermostable proteins
have a lower percentage of alpha helices compared to beta sheets, since beta sheets can hold the tertiary
structure together more effectively than alpha helices.

A fold family contains proteins that have the same major secondary structures in the same arrangement
with the same topological connections and are clearly related by evolution. Folds are formed because of
thermodynamic stability. Structural domains are physically independent regions of the tertiary
structure, which have a specific function. Proteins may have common domains even if their overall
tertiary structures and their overall functions are different.

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Procedure for Bioinformatics Analysis of Amylase Sequences
To complete the Bioinformatics assignment below

1. NCBI: To obtain amino acid sequences of all three amylases (used in the enzyme lab) in FASTA

format from the NCBI database using accession numbers: http://www.ncbi.nlm.nih.gov/
Select the Protein database from the dropdown menu. Type the NCBI accession number (without the
GI) into the search box. On the output screen, select FASTA for the single-letter version of the amino
acid sequence. Copy the amino acid sequence, including the heading but simplify it to “>human
amylase”, etc. Paste all 3 FASTA sequences into a Word file.

2. PEPSTATS: To compute statistics, e.g., total number of amino acids, number of polar and nonpolar
amino acids, percentages polar/nonpolar amino acids of each amylase using the amino acid sequences
(from the NCBI database): http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/
Paste the FASTA format amino acid sequence obtained from NCBI into the query box. Do not include
the heading. Complete Table 1. Note: “residues” = amino

acids

3. CLUSTAL OMEGA: To compare amino acid sequences from all 3 amylases and find out percent
identity between the amylases and to understand the differences between homology, identity and
similarity: https://www.ebi.ac.uk/Tools/msa/clustalo/
Copy and paste all three FASTA sequences into the query box. Include the heading as well as the
“>”. Eliminate all extra line spaces between FASTA sequences. Click “Submit”. For proper
alignment, you may need to widen the margins in Word after you paste the results. To complete Table
2 select the Results Summary tab and the link for “percent identity matrix”.

4. BOXSHADE: To highlight (for ease of location) specific amino acid(s) that are conserved in all three
enzymes using the output from the CLUSTAL OMEGA site:
http://www.ch.embnet.org/software/BOX_form.html
Paste the entire results of the Clustal Omega analysis including the “Clustal” heading into the query
box. Change the Output to “RTF-NEW”, the Fraction to “1.0”, and the Input to “Other”. Click on
“Run BOXSHADE”, and you will be taken to a result page where you will find a link to your
alignment editing output. Click on that link and your edited alignment file will be downloaded to your
computer as an MS Word-compatible file.

5. PDB: To look at computer-generated models of the overall 3D structures of individual amylases and
the active site and locate secondary structure motifs, e.g., alpha helices, beta pleated sheets, presence
of cofactor, Ca. Use PDB ID at this site. http://www.rcsb.org/pdb/home/home.do. Copy each
enzyme’s PDB ID from Table 4, and paste it into the PBD website’s search field; each protein’s 3D
structure will appear in a panel on the left. You can copy the image and paste it into your assignment.
Click on the tab “Sequence”, above the 3D structure, to obtain the percent alpha helix and beta sheet
content of each amylase.

http://www.ncbi.nlm.nih.gov/

http://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/

https://www.ebi.ac.uk/Tools/msa/clustalo/

http://www.ch.embnet.org/software/BOX_form.html

http://www.rcsb.org/pdb/home/home.do

18

BIOINFORMATICS ASSIGNMENT

1. Complete Table 1 after using the PEPSTATS tool:

Alpha amylase

Total
Number of

amino acids

Number of
polar amino

acids

% of polar
amino acids

Number of
non-polar

amino acids

% of
non-polar

amino acids

Homo sapiens
(salivary)

Aspergillus
oryzae

Geobacillus
stearothermophilus

2. Complete Table 2 after obtaining the Percent Identity Matrix with CLUSTAL OMEGA:

Source of
alpha amylase

Homo sapiens
(salivary)
Aspergillus
oryzae
Geobacillus
stearothermophilus
Homo sapiens
(salivary)

Aspergillus
oryzae

Geobacillus
stearothermophilus

3. In human salivary amylase, the amino acids that make up the active site are aspartate197 (D197),
glutamate233 (E233) and aspartate300 (D300) (the numbers indicate the order of these amino acids
in the human sequence). Using the BOXSHADE alignment, determine if these amino acids are
conserved in the fungal and bacterial amylases.

4. What secondary structures do you recognize? What is the importance of secondary structures?

19

5. Using the PDB website, enter the percent alpha helix and beta sheet content for all three amylase
enzymes, in Table 3 below.

Human Fungal Bacterial

% Alpha helices

% Beta sheets

6. Discuss the differences in alpha helix and beta sheet content between the three amylases. What do
these differences signify?

7. Obtain the 3D structures of all three amylases, and attach them to your assignment. Draw arrows
and label an alpha helix and a beta sheet on each structure.

20

APPENDIX

Data from the study of the Effect of Temperature on Human Amylase Activity

Temperature ( 0C) Absorbance
at 540nm

Maltose produced
(mg/ml)

Amylase Activity
(mg/ml/min)

0 0.107

25 0.568

37 0.874

45 0.342

65 0.031

85 0.008

Data from the Study of the Effect of pH on Human Amylase Activity

pH Absorbance
at 540nm

Maltose produced
(mg/ml)
Amylase Activity
(mg/ml/min)

1 0.029

3 0.103

5 0.267

7 0.945

9 0.112

12 0.015

21

Data from the study of the Effect of Temperature on Fungal Amylase Activity

Temperature ( 0C) Absorbance
at 540nm
Maltose produced
(mg/ml)
Amylase Activity
(mg/ml/min)

0 0.129

25 0.246

37 0.539

45 0.839

65 0.137

85 0.024

Data from the Study of the Effect of pH on Fungal Amylase Activity

pH Absorbance
at 540nm
Maltose produced
(mg/ml)
Amylase Activity
(mg/ml/min)

1 0.119

3 0.231

5 0.945

7 0.367

9 0.091

12 0.016

22

Data from the study of the Effect of Temperature on Bacterial Amylase Activity

Temperature ( 0C) Absorbance
at 540nm
Maltose produced
(mg/ml)
Amylase Activity
(mg/ml/min)

0 0.116

25 0.237

37 0.309

45 0.391

65 0.895

85 1.029

Data from the Study of the Effect of pH on Bacterial Amylase Activity

pH Absorbance
at 540nm
Maltose produced
(mg/ml)
Amylase Activity
(mg/ml/min)

1 0.107

3 0.209

5 0.637

7 0.995

9 0.048

12 0.037

EXERCISE 4
ENZYMES
Functional and Structural Analysis
OBJECTIVES
Factors that affect the rate of enzyme activity:
PART II: STANDARD CURVE FOR ESTIMATION OF MALTOSE
Part III: Test your hypothesis