Aaron Gardner · Wilko Duprez

Sarah Stauffer · Dewi Ayu Kencana Ungu

Frederik Clauson-Kaas

Labster Virtual
Lab Experiments
Basic Biochemistry
Labster Virtual Lab Experiments:
Basic Biochemistry
Aaron Gardner  Wilko Duprez 
Sarah Stauffer 
Dewi Ayu Kencana Ungu 
Frederik Clauson-Kaas

Labster Virtual Lab

Experiments:
Basic Biochemistry
Aaron Gardner Dewi Ayu Kencana Ungu
Labster Group ApS Labster Group ApS
København K, Denmark København K, Denmark

Wilko Duprez Frederik Clauson-Kaas

Labster Group ApS Labster Group ApS
København K, Denmark København K, Denmark

Sarah Stauffer
Labster Group ApS
København K, Denmark

ISBN 978-3-662-58498-9 ISBN 978-3-662-58499-6 (eBook)

https://doi.org/10.1007/978-3-662-58499-6

Springer Spektrum
© Labster ApS under license to Springer Verlag GmbH 2019
This work is subject to copyright. All rights are reserved by © Labster ApS under license to
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Preface

Welcome to the “Basic Biochemistry” textbook, which is part of the “Labster Vir-
tual Lab Experiments” series.
This book will help you to learn the key concepts of basic biochemistry while
applying your newly acquired knowledge in a virtual lab environment. In each
chapter you will be introduced to one of four virtual lab simulations and the true-
to-life missions that you will encounter when playing the simulations. Study the
theory section presented in each of the chapters closely and you will be fully pre-
pared to master the challenging tasks in the virtual lab!
Finally, you will find learning objectives and techniques covered by the virtual
lab simulation at the end of each chapter to easily align its content with your exam
preparation.

About Labster

Labster is a company dedicated to developing virtual lab simulations that are de-
signed to stimulate students’ natural curiosity and highlight the connection be-
tween science and the real world. These simulations have been shown to improve
the achievement of learning outcomes among students, by making the learning
experience more immersive and engaging. The content of this book was created
by the Labster team members Dr. Aaron Gardner, Dr. Wilko Duprez, Dewi Ayu
Kencana Ungu, Dr. Sarah Stauffer, Dr. Frederik Clauson-Kaas, and Silvia Tjong.

About This Textbook

You may think of biology and chemistry as distinct subjects. In fact, the two sub-
jects are inextricably linked, with many key biological processes being based on
v
vi Preface

chemical equations. Biochemistry is the field of study formed when biology and
chemistry meet and covers everything from the molecular interactions of DNA all
the way through to regulation and modulation of our dietary intake. With an under-
standing of biochemistry you can then explore the ever more complex structures,
functions and processes that regulate life.
In the first chapter of this volume, we’ll start with the basics and give an intro-
duction to chemical bonds showing how small changes can have big effects. Newly
equipped with this knowledge you’ll embark on a biochemical adventure, studying
the building blocks of our food and drink before learning how our bodies process
them.

Ionic and Covalent Bonds

If we know that a lot of biological processes rely on chemistry, we should first look
at some key chemical properties. In the Ionic and Covalent Bonds simulation you’ll
investigate how the type and number of chemical bonds formed between atoms can
significantly alter the structure and function of molecules and the macromolecules
they form. You will also apply a variety of biochemical techniques to identify
different substances, which should give you some idea how our body will react to
them. Will you be able to use your new knowledge to help your friend identify two
mysterious substances?

Introduction to Biological Macromolecules

So now you know how atoms bond to form molecules, what happens next? These
individual molecules often bind together to form macromolecules, very large
molecules comprised of many smaller molecules or monomers. These macro-
molecules are vital to life, forming the basic structure of cells, acting as the storage
mechanism for genetic information and transmitting information around the body.
In the Introduction to Biological Macromolecules simulation, you will focus on
the source of many of these macromolecules, our food, performing a variety of
biochemical assays in order to detect their presence and identifying which foods
are rich in which macromolecules. Finally, you will pull all of your research
together and discuss what makes a healthy diet with your friend!

Carbohydrates
The many forms of carbohydrates in our diet represent an essential energy source,
but how our body deals with these carbohydrates varies widely. In the Carbohy-
drate simulation you will be able to observe at the molecular level how soluble and
insoluble carbohydrates are digested and utilized as energy and measure our virtual
Preface vii

subject’s blood glucose level to see what impact they have. Will you be able to use
your carbohydrate knowledge to help your friend prepare for a long distance run?

Enzyme Kinetics
Chemical reactions often look straightforward on paper, but in real life often rely
on catalysts to promote and increase the reaction rate. Enzymes are biological cat-
alysts, driving key reactions without being used up in the process. Importantly,
enzyme activity is highly affected by small changes in their structure or environ-
ment. In the Enzyme Kinetics simulation you will be able to observe these effects
at the molecular level and see how mutations in the alcohol dehydrogenase gene
can drastically affect our ability to metabolize alcohol byproducts.

How to access the virtual lab simulations?

You can access the four virtual lab simulations included in this book at www.
labster.com/springer.
If you have purchased a printed copy of this textbook, you will find a voucher
code on the last page, which gives you free access to the four simulations for the
duration of one semester (six months).
If you are using the e-book version, you can sign up and buy access to the
simulations through the same link.
Please be aware that the six month period starts once you sign in for the first
time.
If you have any questions about the use of the voucher, you can contact us at
[email protected].
Contents

1 Ionic and Covalent Bonds . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.1 Ionic and Covalent Bonds Simulation . . . . . . . . . . . . . . . . . 2
1.2 Ionic and Covalent Bonds Theory Content . . . . . . . . . . . . . . 4
1.3 Let’s Get Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2 Introduction to Biological Macromolecules . . . . . . . . . . . . . . . 19

2.1 Introduction to Biological Macromolecules Simulation . . . . . . 20
2.2 Introduction to Biological Macromolecules Theory Content . . . 22
2.3 Let’s Get Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

3 Carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.1 Carbohydrate Simulation . . . . . . . . . . . . . . . . . . . . . . . . 44
3.2 Carbohydrates Theory Content . . . . . . . . . . . . . . . . . . . . . 46
3.3 Let’s Get Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

4 Enzyme Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
4.1 Enzyme Kinetics Simulation . . . . . . . . . . . . . . . . . . . . . . 58
4.2 Enzyme Kinetics Theory Content . . . . . . . . . . . . . . . . . . . 60
4.3 Let’s Get Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Further Reading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

ix
Ionic and Covalent Bonds
1

© Labster ApS under license to Springer Verlag GmbH 2019 1

A. Gardner et al., Labster Virtual Lab Experiments: Basic Biochemistry,
https://doi.org/10.1007/978-3-662-58499-6_1
2 1 Ionic and Covalent Bonds

1.1 Ionic and Covalent Bonds Simulation

Have you ever wondered how everything around you is held together? Well, in
this simulation, you will learn the basics of atomic bonding in ionic and covalent
compounds and how to distinguish those compounds experimentally. Using this
knowledge you’ll help your friend to analyze two mysterious substances he re-
ceived from an alchemist. By testing various physical properties you will be able
to narrow down and eventually identify the substances in question and see if the
alchemist is telling the truth.

Test the Solubility and Conductivity of Samples

Atoms can interact in many different ways, giving a compound specific properties.
In the first mission of the Ionic and Covalent Bonds simulation, your task is to
identify the appropriate laboratory equipment to test the solubility and conductivity
of the two substances when dissolved in water (Fig. 1.1). You will then explore
how these properties differ between ionic and covalent compounds.

Draw Lewis Dot Structures

In the second part of the Ionic and Covalent Bonds simulation, you will learn about
the octet rule and how to apply this to building Lewis dot structures in a virtual

Fig. 1.1 Testing the solubility and conductivity of samples in the Ionic and Covalent Bonds
simulation
1.1 Ionic and Covalent Bonds Simulation 3

Fig. 1.2 Learn about and draw your own Lewis dot structures in the Ionic and Covalent
Bonds simulation

Fig. 1.3 Determine the melting point of two unknown substances in the Ionic and Covalent
Bonds simulation
4 1 Ionic and Covalent Bonds

drawing activity (Fig. 1.2). You will realize that there are many ways to form
covalent bonds, depending on the atoms involved and their electron configuration,
and how small changes in structure can have big effects on function.

Determine Melting Points

In the last mission, your task is to determine the melting point of the two substances
by using melting point apparatus. You will explore how your results are connected
to the ability of ions to form a lattice structure (Fig. 1.3).

1.2 Ionic and Covalent Bonds Theory Content

In the Ionic and Covalent Bonds simulation we’re going to delve inside individual
molecules to see how they are bound together. You will learn how the types of
bonds present in molecules can significantly impact their structure and activity,
before practicing several key experimental techniques which can be used to analyze
and describe the characteristics of unknown substances. Will you be able to use
these techniques to help your friend to identify two unknown substances he’s been
given?

Periodic Table

The periodic table is an arrangement of all chemical elements, sorted by their

atomic number. Rows within the table are termed “periods” (hence, periodic ta-
ble), whereas columns are known as groups. The table is structured in such a way
as to inform about electron configuration and chemical properties based on group
and period number. For example, the elements in group 18 are known as the noble
gases, and they share a range of chemical properties.
It is also possible to further divide the table into blocks depending on the last
populated electron shell, an idea we will cover later in the theory content (Fig. 1.4).

Fig. 1.4 The periodic table. The periodic table is an arrangement of all chemical elements,
sorted by their atomic number. You can use the above table as a reference throughout this
chapter
I
1.2 Ionic and Covalent Bonds Theory Content 5
6 1 Ionic and Covalent Bonds

Fig. 1.5 Atomic number as they appear on the periodic table. Each element is repre-
sented by a symbol in the periodic table, in this case K, which represents potassium. The
atomic number defines the number of protons in the atomic nucleus of this element

Atomic number

The atomic number of a specific chemical element indicates the number of protons
in its nucleus and is identical to the charge number in the nucleus. In the neutral
state of an atom, the atomic number also equals the number of electrons (Fig. 1.5).
The atomic number can be added to the number of neutrons in an element to
give the mass number; the mass of electrons is negligible and is so not counted.

Electron Shell

Within an atom, protons, and neutrons form the dense nucleus, whereas the elec-
trons orbit the nucleus in shells, with each shell consisting of subshells. The shell
nearest the nucleus is named K, with subsequent shells named alphabetically, for
example, K, L, M, N . . .
Subshells exist within shells, with each subshell able to host a defined number
of electrons. These subshells are named s, p, d, f, and g. The number of subshells
present within a shell increases as they move away from the nucleus. For example,
the K shell contains only an s subshell, whereas the L shell contains both an s and
a p subshell. As electrons are attracted to the positively charged nucleus, shells are
generally filled in order moving away from the nucleus. Possible permutations are
detailed in Table 1.1 below.
The outermost shell of an atom with any electrons in it is termed the valence
shell, and the number of electrons in this shell determines the chemical properties
of the element. For example, the noble gases which have full valence shells are
considered to be mostly unreactive with other elements.
1.2 Ionic and Covalent Bonds Theory Content 7

Table 1.1 The various electron shells and subshells. Electrons are arranged in shells ex-
panding outwards from the atom’s nucleus. They are named K, L, M, N . . . and onwards.
Shells also contain subshells termed s, p, d, and f. The first shell contains only one subshell,
the second two and so on
Shell Subshell Maximum # of subshell electrons Maximum # of shell electrons
K 1s 2 2
L 2s 2 8
2p 6
M 3s 2 18
3p 6
3d 10
N 4s 2 32
4p 6
4d 10
4f 14

Ions

When the number of electrons and protons in an atom or molecule don’t match,
they are referred to as ions. The word ion comes from the Greek word “ἰó”,
meaning “to go”, and was so named by the physicist Michael Faraday for its ability
to carry a charge between electrodes in an aqueous solution. Although at the time
Faraday was unaware of the character of these ions, it is now possible to predict
the electrical charge of elements and compounds based on their positions in the
periodic table.
Faraday also identified the two types of ions, naming them anions and cations,
depending on their ionic charge.

Anions

Negatively charged ions are called anions. The negative charge is a result of having
more electrons than protons, as can be seen in the example of chloride (Cl ) in
Fig. 1.6 below. The minus ( ) symbol denotes that the chlorine atom has gained
one electron forming a chloride ion. Another example would be sulfide (S2 ), in
this instance the (2 ) denotes that the sulfur atom has gained two electrons, forming
a sulfide ion.
8 1 Ionic and Covalent Bonds

Fig. 1.6 A chloride anion. A chlorine (Cl) atom has an equal number of protons and
electrons (17) and is hence uncharged. In contrast, a chloride anion (Cl) has gained one
electron. This results in more electrons than protons and gives Cl an overall negative charge
of 1. The charge is indicated by a superscript 

Cations

Positively charged ions are called cations. The negative charge is a result of having
fewer electrons than protons, as can be seen in the example of potassium (K+ ) in
Fig. 1.7 below. The plus (+ ) symbol denotes that the potassium atom has lost one
electron forming a potassium ion. Another example would be calcium (Ca2+ ), in
this instance the (2+ ) denotes that the calcium atom has lost two electrons forming
a calcium ion.

Ionic Charge

One can predict the charge of an ion by checking the location of the atom in the
periodic table. Atoms of many main group metals and non-metals tend to lose or
gain enough electrons to obtain the same number of electrons as an atom of the
noble gases.
1.2 Ionic and Covalent Bonds Theory Content 9

Fig. 1.7 A potassium cation. A potassium (K) atom has an equal number of protons and
electrons (19) and is, hence, uncharged. In contrast, a potassium cation (K+ ) has lost one
electron. This results in fewer electrons than protons and gives K an overall positive charge
of C1. The charge is indicated by a superscript +

For example, atoms in groups 1 and 2 tend to lose electrons to achieve the same
number of electrons as the preceding noble gas atom. Using our examples above,
potassium loses one electron and calcium loses two, giving both the same electron
number as argon.
In contrast, atoms in group 14–17 tend to gain additional electrons to acquire
the same electron configuration as the subsequent noble gas in the period. Again
using the above example, chlorine gains one electron and sulfur two, giving both
the same electron number as argon.

Valence Electron

The outermost shell of an atom is termed the valence shell. Electrons within this
shell are called valence electrons, and their number determines the chemical prop-
erties of an element and how it reacts when forming chemical bonds.
For many elements, the valence electron number can be determined from the
periodic table. For groups 1 and 2, the valence electron number simply matches the
10 1 Ionic and Covalent Bonds

group number. For groups 13–18, the valence electron number is the group number
minus 10. Unfortunately, for groups 3–12, there is no simple way to determine the
valence electron number using the periodic table.

Chemical Bonds

Within a chemical compound, atoms are held together by chemical bonds. There
are three types of chemical bonds; ionic, covalent and metallic, with the first two
being the focus of this chapter and simulation. The bond is either established by
an electrostatic force of attraction between ions of opposite charges or by sharing
electrons. For example, Na+ and Cl could bond forming the compound NaCl,
which has an overall neutral charge. The overall aim of sharing electrons is to
stabilize the valence shell by allowing each component to “fill” its valence shell.
For example, two chlorine atoms can form a bond by each sharing an electron
with each other, and thus both atoms achieve a filled valence shell, forming the
compound Cl2 .

Octet Rule

The tendency for atoms of most common elements to form chemical bonds in such
a way that each atom obtains eight valence electrons is described by the “octet
rule”. Eight electrons in the valence shell is a particularly stable state with the
same electronic configuration as the inert noble gases.
Two notable exceptions to the rule are hydrogen and lithium. Hydrogen only
needs to gain one additional electron, and lithium needs only to lose one electron
to attain a stable configuration with two electrons. This is because they have very
few electrons, and they aim towards the electron configuration of the noble gas
Helium, which only has two electrons in its K valence shell (Table 1.1).

Ionic Bonding

Ionic bonds are formed between ions and held together by the electrical attrac-
tion of opposite charges, as for example between the metal sodium and non-metal
chloride in sodium chloride (Na+ C Cl ! NaCl) or in potassium chloride (K+ C
Cl ! KCl). Ionic bonding leads to the formation of an ionic or crystal lattice.
When a cation forms an ionic bond with an anion, the number of negative charges
1.2 Ionic and Covalent Bonds Theory Content 11

Fig. 1.8 An ionic bond between a potassium (K+ ) ion and chloride ion (Cl). The
positively charged potassium cation and negatively charged chloride anion form a bond,
canceling out each other’s charge, forming a neutral KCl molecule

is typically equal to the number of positive charges, and thus an ionic compound
has an overall neutral charge (Fig. 1.8).

Ionic Compounds

A compound that is composed of cations and anions and is held together by an

ionic bond is called an ionic compound. An ionic compound is overall electrically
neutral and typically formed between metals and non-metals.
Examples of ionic compounds include:

 NaBr – sodium bromide

 KBr – potassium bromide
 NaCl – sodium chloride (commonly known as table salt)
 NaF – sodium fluoride
 KI – potassium iodide
 KCl – potassium chloride
 CaCl2 – calcium chloride
 K2 O – potassium oxide
 MgO – magnesium oxide

Ionic Compound Characteristics

Many ionic compounds are highly soluble in water. This means they dissociate
into their component ions when dissolved in water. The charged ions interact with
12 1 Ionic and Covalent Bonds

Fig. 1.9 KCl dissolved in water. When dissolved in water, the charged chloride (red) and
potassium (blue) ions interact with either the positively charged hydrogen (grey) or nega-
tively charged oxygen (turquoise) atoms, which form a water (H2 O) molecule

the partially negative oxygen and partially positive hydrogen in H2 O (Fig. 1.9).
However, they are typically insoluble in less polar solvents such as ethanol.
Ionic compounds are not electrically conductive in their solid form. However,
when dissolved in water, anions and cations are released into the solution and fa-
cilitate the flow of electricity.
Most ionic substances are solid at room temperature, forming a tight crystal
lattice type structure. Due to the high strength of the bonds they typically have
high melting and boiling points.

Covalent Bonding

Non-metals form covalent, or molecular, bonds by sharing electrons equally be-

tween each other. For most atoms, the sharing of electrons in a covalent bond
allows them to obtain a stable electron configuration, following the octet rule. If
the electrons are not shared equally, it is called a polar covalent bond.

Covalent Compounds

A covalent compound, sometimes also called a molecular compound, consists of

discrete neutral atoms that are connected by covalent bonds. They are formed
solely between non-metals. All organic compounds, including carbohydrates,
1.2 Ionic and Covalent Bonds Theory Content 13

lipids, proteins and nucleic acids, are examples of covalent compounds. Other
common compounds include:

 CO2 – carbon dioxide

 H2 O – water
 CH4 – methane
 HCl – hydrogen chloride
 NH3 – ammonia
 C6 H12 O6 – glucose

Covalent Compound Characteristics

Many covalent compounds are less soluble in water than ionic compounds, but
they are often more soluble in organic solvents such as ethanol.
Due to their lack of charge covalent compounds do not conduct electricity in
water.
Most covalent compounds form single molecules with weak interactions be-
tween them, meaning they do not form the strong crystal lattices associated with
ionic compounds. As such, their melting and boiling points are much lower, and
most compounds exist as liquids or gases at room temperature. Exceptions include
larger covalent molecules ranging from glucose all the way up to extremely large
proteins and nucleic acid molecules.

Caffeine

In the Ionic and Covalent Bonds simulation we use caffeine as a control compound.
Caffeine is a stimulant, which acts upon the central nervous system (CNS), acting
to keep us awake and more alert. Caffeine belongs to a group of substances called
alkaloids and is a covalent compound. Alkaloids are found naturally in several
plant species, such as coffee, tea, and cocoa.

Physical properties

A physical property is any measurable property of a substance or material that is

not associated with a change in chemical composition. A physical property can
either be determined without affecting the object, such as density, hardness, and
14 1 Ionic and Covalent Bonds

color or measured following a change of the physical state of the matter, such as
melting point, boiling point, electrical conductivity, and solubility.

Solubility

Solubility is a physical property describing the ability of a chemical substance,

called a solute, to dissolve in another chemical substance, called a solvent.
The solubility of a chemical depends on the polarity of the solvent (for example,
water) compared with that of the solute, and the amount of energy it takes to break
the intermolecular forces of the solute. For example, because it takes a lot of
energy to break metallic bonds, metals are insoluble in water. As mentioned earlier,
covalent compounds can often be dissolved in organic solvents.

Electrolytic Conductivity

An aqueous solution that can conduct an electric current is called conductive. The
unit of electrolytic conductivity is siemens per meter (S/m). Conductive solutions
can include salts (ionic compounds) or some acids dissolved in water, where the
ions are free to move.
Water itself is not electrically conductive. However, when an ionic compound is
dissolved in water, it dissociates into cations and anions, enabling a flow of electric
charges through the solution.

Phase Changes

Heating a solid substance will increase the energy of its atoms or ions up to a point
where the energy levels become high enough to partially disrupt the forces holding
the atoms or ions in their fixed position. At this point, the solid starts to transition
into the liquid state, which is called melting. The temperature at which the solid
and liquid phase are in equilibrium is called the melting point.
The melting point depends on the attractive forces between the atoms or ions in
the solid—the stronger the attractive forces, the higher the melting point.
Upon further heating liquids will reach the highest temperature of this state, the
boiling point, to become gases, where the constituents have very little interaction
with each other. When gases are subjected to extreme heat or electromagnetic
1.2 Ionic and Covalent Bonds Theory Content 15

forces, electrons are ripped from the outer electron shell as the atoms transition
into the plasma state.

Lewis Dot Structures

Forming a chemical bond (ionic or covalent) involves either the electrostatic at-
traction of oppositely charged ions, where a metal “donates” electron(s) to a non-
metal, or the sharing of electron(s). One way to visualize valence shell electrons
in a chemical bond is the use of Lewis dot structures. This concept was introduced
by Gilbert N. Lewis in 1916.
In Lewis structures, every valence shell electron is illustrated as a single dot.
Lone electron pairs are shown as two dots around the element symbol. Sometimes
a line is used to indicate a shared or lone pair of electrons.
According to the Lewis structure concept, the formation of an ionic bond be-
tween potassium (K) and chlorine (Cl), and the covalent bond between two chlo-
rines, can be visualized as shown below in Fig. 1.10.

How to Draw Lewis Structures

Follow the step-by-step guide to draw Lewis structures for various compounds.

Step 1 Count all valence (outer shell) electrons.

Step 2 Draw the least electronegative atom in the center and arrange the other
atoms around the central atom.

Fig. 1.10 Lewis dot structures for ionic and covalent compounds. A) The Lewis dot
structure for the ionic compound KCl is shown. K donates one of its electrons to Cl to
provide filled valence shells. This then forms a bond between the K+ and Cl ions. B) Two
chlorine atoms each share an electron with each other to form a stable Cl2 molecule, where
each has a filled valence shell
16 1 Ionic and Covalent Bonds

Step 3 Connect each atom to the central atom with a single bond (one electron
pair).

Step 4 Complete octets with electrons as lone pairs at the terminal atoms first
(except for hydrogen). Add remaining atoms (if any) to the central atom.

Step 5 Complete the central atom’s octet by making multiple bonds with the ter-
minal atoms.

Step 6 (for some molecules) Depending on which electrons you “moved” in

step 5, a variety of resonance structures can be determined.

Lewis Dot Structure of CO2

Let’s see how to apply the five steps to the Lewis structure of carbon dioxide or
short CO2 – a gas that we breathe every day (Fig. 1.11).

Step 1 The total number of valence electrons in CO2 is 4.C/C6.O/C6.O/ D 16.

Step 2 C 3 Arrange the atoms and connect the central atom with a single bond.
Subtract those electrons from the total number of electrons.

Step 4 Now complete octets with electrons as lone pairs at the terminal atoms first
and subtract those electrons from the total number of electrons.

Step 5 Finally, complete the central atom’s octet by making multiple bonds with
the terminal atoms. Lone electron pairs can also be shown as a single line.

Step 6 can be omitted in the case of CO2 , as only one possible Lewis dot structure
exists.

Fig. 1.11 Lewis dot structures for carbon dioxide CO2 . The Lewis dot structure for the co-
valent compound CO2 is shown above. Each carbon and oxygen atom shares two electrons,
allowing them to form a stable molecule where each atom has a filled valence shell
Further Reading 17

1.3 Let’s Get Started

Now you know the difference between ionic and covalent substances and the bonds
that form them. Join your friend on a quest to analyze the two unknown substances
he got from an alchemist to cure his migraine and put your knowledge to the test
in the Ionic and Covalent Bonds simulation.

Techniques Used in the Lab

 Conductivity measurement
 Melting point determination

Learning Objectives
At the end of this simulation, you will be able to . . .

 Describe the formation of ionic and covalent bonds

 Identify anions and cations
 Apply the octet rule
 Describe ionic lattice structure
 Draw Lewis dot structures
 Explain the formation of single, double, and triple bonds
 Distinguish between ionic compounds and covalent compounds

ACCESS THE VIRTUAL LAB SIMULATION HERE www.labster.com/

springer BY USING THE UNIQUE CODE AT THE END OF THE PRINTED
BOOK. IF YOU USE THE E-BOOK, YOU CAN PURCHASE ACCESS TO
THE SIMULATIONS THROUGH THE SAME LINK.

Further Reading
OpenStax CNX (2018) OpenStax, chemistry. http://cnx.org/contents/85abf193-2bd2-4908-
[email protected]. Accessed 2 Aug 2018
Brown TL (2015) Chemistry: the central science. Pearson, New York
Introduction to Biological Macromolecules
2

© Labster ApS under license to Springer Verlag GmbH 2019 19

A. Gardner et al., Labster Virtual Lab Experiments: Basic Biochemistry,
https://doi.org/10.1007/978-3-662-58499-6_2
20 2 Introduction to Biological Macromolecules

2.1 Introduction to Biological Macromolecules Simulation

As you’ve just learned about how atoms bond together to form compounds, the
next step is to look at the huge variety of biological macromolecules that these
compounds can give rise to. Biological macromolecules are very large molecules
created by the polymerization of small units called monomers. These macro-
molecules are a great source of energy and building materials for our body, which
we gain through our food. In this simulation, you will learn about the wide va-
riety of macromolecules and their structure and function, before pulling all this
information together discuss healthy diets with your friend.

Learn the Basics about Biological Macromolecules

There are several types of biological macromolecules, namely: carbohydrates, pro-
teins, lipids, and nucleic acids. Each has its own unique structure and function, but
all share some common naming conventions. In this simulation, you will learn
about these naming conventions and apply them to relevant examples from daily
life (Fig. 2.1).

Fig. 2.1 Learn about the basic characteristics of important macromolecules from daily life
in the Introduction to Biological Macromolecules simulation
2.1 Introduction to Biological Macromolecules Simulation 21

Observe Macromolecules at the Molecular Level

Understanding how macromolecules are constructed, and how small changes in
their structure can have big effects on their function, is of key importance in un-
derstanding biochemistry. Using the power of the virtual lab you will be able to
observe the formation and structure of macromolecules at the molecular level, giv-
ing a unique view into their formation (Fig. 2.2).

Use Your Knowledge of Macromolecules in a Discussion with Your Friend

While knowledge is in itself a wonderful thing, it is the application of that knowl-
edge that will make you a true scientist. In the Introduction to Biological Macro-
molecules simulation, you will investigate the types of macromolecules found in
common foods by performing a series of biochemical tests. Can you then use your
knowledge of foods and macromolecules to discuss healthy diets with your friend
(Fig. 2.3)?

Fig. 2.2 In the Introduction to Biological Macromolecules simulation you will be able to
observe the structure and formation of macromolecules at the molecular level
22 2 Introduction to Biological Macromolecules

Fig. 2.3 Can you discuss with your friend how to follow a healthy diet in the Introduction
to Biological Macromolecules simulation?

2.2 Introduction to Biological Macromolecules

Theory Content

The topics covered by the Introduction to Biological Macromolecules Theory Con-

tent address all the concepts discussed in the associated simulation and will give
you a good understanding of how vital the study of biochemistry is to our daily
lives. You will see this in full effect as you discuss how to improve your diet with
your friend.

Biological Macromolecules

Macromolecules are very large molecules, such as deoxyribose nucleic acid

(DNA), created by the polymerization of smaller molecules known as monomers,
nucleic acids in the case of DNA. Macromolecules are widely used throughout
our body but are also typically our most common source of energy and cellular
building materials, which we get from the food we eat.
2.2 Introduction to Biological Macromolecules Theory Content 23

Table 2.1 Example macromolecules for the various families discussed in the Introduction
to Biological Macromolecules simulation
Macromolecule Family Type Specific type
DNA Nucleic acid Polymer DNA polymer
siRNAa Nucleic acid Oligomer RNA oligomer
Starch Carbohydrate Polymer Polysaccharide
Sucrose Carbohydrate Dimer Disaccharide
Glucose Carbohydrate Monomer Monosaccharide
Glucagon Protein Polymer Peptide
Glycine Protein Monomer Amino acid
Fatty acid Lipid Monomer Carboxylic acid

a
Small interfering ribonucleic acid

There are several types of biological macromolecules, which are detailed below
in Table 2.1:

 Carbohydrates
 Proteins
 Lipids
 Nucleic acids

All macromolecules, with the exception of lipids, are polymers. A polymer is

a long molecule composed of chains of monomers linked together.
The terms polymer and macromolecule are typically used to refer to extremely
long chains of monomers. So, when we want to refer to shorter chains we use
the term oligomer. For very short chain molecules, we can specify the length, for
example dimers are comprised of two molecules, whereas trimers are derived from
three monomers.
In biochemistry, an oligomer usually refers to a macromolecular complex
formed by non-covalent bonding of a few macromolecules, such as short nucleic
acids or small proteins.

Carbohydrates

Carbohydrates are macromolecules built from sugars. They can exist as sim-
ple, single sugar molecules (monosaccharides), or chains of two or more sugar
24 2 Introduction to Biological Macromolecules

molecules (disaccharides and polysaccharides). Carbohydrates are an important

source of energy and structural material for organisms.
We focus on carbohydrates in the third simulation, and so provide a more in-
depth overview in the associated chapter.

Monosaccharides

Monosaccharides (mono = “one”, sacchar = “sweet”) are simple sugars. In

monosaccharides, the number of carbon atoms usually ranges from three to seven.
Most monosaccharide names end with the suffix “-ose”. If the sugar has an
aldehyde group (the functional group with the structure R-CHO), it is known as
an aldose, and if it has a ketone group (the functional group with the structure

Fig. 2.4 Various chain length monosaccharides. Here, three (glyceraldehyde), five (ri-
bose), and six (glucose) carbon monosaccharides are shown. They can also be referred to as
triose, pentose, and hexose based on the length of the carbon backbone
2.2 Introduction to Biological Macromolecules Theory Content 25

RC(=O)R0 ), it is known as a ketose. Depending on the number of carbons in the

sugar, they also may be known as trioses (three carbons), pentoses (five carbons),
and/or hexoses (six carbons) (Fig. 2.4).
The most common monosaccharide is glucose, the building block of many
important carbohydrates. Galactose, part of lactose or milk sugar, and fructose,
found in sucrose in fruits, are other common monosaccharides. Although glu-
cose, galactose, and fructose all have the same chemical formula (C6 H12 O6 ), they
differ structurally and chemically and are known as isomers, because of the dif-
ferent arrangement of functional groups around the asymmetric carbon (Fig. 2.5).
Monosaccharides can exist as a linear chain or as ring-shaped molecules, which is
visualized in the Introduction to Biological Macromolecules simulation.

Fig. 2.5 Variation in six-carbon monosaccharides. Glucose, galactose, and fructose all
have the same chemical formula (C6 H12 O6 ) but differ structurally and chemically; they are
therefore known as isomers
26 2 Introduction to Biological Macromolecules

Glucose

Glucose is the most common sugar monomer with the chemical formula C6 H12 O6 .
Glucose is an important source of energy required during cellular respiration in
humans and other animals. Energy is released from glucose as it is catabolized,
with that energy used to make adenosine tri-phosphate (ATP). Plants synthesize
glucose using carbon dioxide and water during photosynthesis, and this glucose, in
turn, is used for the energy requirements of the plant. Excess glucose is often stored
as starch, which is catabolized by humans and other animals that feed on plants.

Ring-Straight-Chain Isomerism

Monosaccharides can exist as a linear chain or as a ring-shaped molecule, for ex-

ample, in aqueous solutions they are usually found in the ring form. When glucose
is present in a ring form, it can have two different arrangements of the hydroxyl
group (OH) around the anomeric carbon (carbon 1 that becomes asymmetric in the
process of ring formation).
Both alpha (˛) and beta (ˇ) arrangements are possible; if the hydroxyl group is
located below carbon 1 in the glucose molecule it is said to be in the alpha position,
whereas if it is above the plane, it is said to be in the beta position (Fig. 2.6). This
isomerism alters how the glucose monomers bind with each other producing very
different polysaccharide structures.

Disaccharides

Disaccharides (di = “two”) consist of two sugar molecules. They form when
two monosaccharides undergo a dehydration reaction, forming a glycosidic bond
(Fig. 2.7). These bonds can be of the alpha or the beta type, as explained above.
Some common disaccharides are shown in Table 2.2.

Table 2.2 Common disaccharides. Details of the chemistry and source of three common
disaccharides
Name Consists of Common source
Sucrose Glucose + fructose Table sugar
Maltose Glucose + glucose Malt
Lactose Glucose + galactose Milk
2.2 Introduction to Biological Macromolecules Theory Content 27

Fig. 2.6 Ring-straight chain isomerism. When glucose is formed into a ring, it can be
divided into two isomers, alpha and beta, depending on how the hydroxyl group is orientated.
If below the plane of carbon 1 (the carbon with a double bond to oxygen in the linear form)
it is referred to as alpha glucose, if above the plane then it is known as beta glucose

Glycosidic Bonds

A glycosidic bond is a covalent bond formed between a carbohydrate molecule

and another molecule. In this reaction, the hydroxyl group of the carbohydrate
combines with the hydrogen of another organic molecule, releasing a molecule of
water and forming a covalent bond. Glycosidic bonds can be of the alpha or the
beta type. An alpha-glycosidic bond is formed when both carbons have the same
stereochemistry (Fig. 2.7), whereas a beta-glycosidic bond occurs when the two
carbons have different stereochemistry.
28 2 Introduction to Biological Macromolecules

Fig. 2.7 Formation of sucrose. Sucrose is formed when a glucose and fructose molecule
bind via a glycosidic bond between carbons 1 and 2, respectively. In this example, an alpha
glycosidic bond is formed as both molecules have the same stereochemistry, with the binding
OH groups both located below the plane of carbon 1

Polysaccharides

Polysaccharides are a long chain of monosaccharides linked by glycosidic bonds

(poly- = “many”). The chain may be branched or unbranched, and it may con-
tain different types of monosaccharides. Common polysaccharides include starch,
glycogen, and cellulose.
2.2 Introduction to Biological Macromolecules Theory Content 29

Starch

Starch is the preferred carbohydrate storage mechanism in plants. It is made up of

a mixture of amylose and amylopectin, both polymers of glucose (Fig. 2.8).
Plants synthesize glucose through photosynthesis, any excess glucose beyond
the plant’s immediate energy needs is stored as starch in areas including the roots
and seeds. Starch in the seeds provides food for the plant embryo as it germinates
and is also a source of food for humans and other animals.
However, the structure of starch means that it is difficult for us to digest. It
must first be broken down into digestible sugars using enzymes, such as salivary
amylases. Such enzymes break apart the polysaccharide chain into simpler sugars
such as maltose and glucose. This free glucose can then be absorbed by specialized
cells in the digestive system.
Starch is composed of glucose monomers joined by either alpha 1,4 or alpha 1,6
glycosidic bonds. The numbers 1,4 and 1,6 refer to the carbon number of the two
residues that have joined to form the bond. The way the glucose molecules bond
forms a helical structure.

Glycogen

While plants store glucose as starch, in humans and other vertebrates, glycogen is
the preferred storage form. It is a highly branched molecule made up of glucose
monomers and is usually stored in the liver or muscle cells.
Excess sugar must be stored as glycogen to avoid causing osmotic pressure in
the cells of animals. Whenever blood glucose levels decrease, glycogen is broken
down to release glucose in a process known as glycogenolysis.

Cellulose

Cellulose is another plant polysaccharide and is probably the most abundant natural
biopolymer on earth because it is the main component in plant cell walls. Cellu-
lose provides plants with structural support; hence wood and paper are mostly
composed of cellulose. Cellulose is made up of glucose monomers that are linked
by beta 1,4 glycosidic bonds. Its structure is similar to that of amylose shown in
Fig. 2.8, however, each second glucose molecule is flipped due to the beta rather
than alpha bonds present. This results in a linear, fibrous structure, as opposed
30 2 Introduction to Biological Macromolecules

Fig. 2.8 Structures of amylose and amylopectin. A) amylose is composed of glucose

monomers that are joined by alpha 1,4 glycosidic bonds forming a linear structure. B) amy-
lopectin is formed from glucose monomers joined by alpha 1,4 or alpha 1,6 glycosidic bonds
giving rise to a branched structure
2.2 Introduction to Biological Macromolecules Theory Content 31

to the helical starch, giving cellulose its rigidity and high tensile strength, a key
requirement for plant cells.
Human digestive enzymes cannot break down the beta 1,4 glycosidic bonds.
However, herbivores such as cows, koalas, buffalos, and horses possess specialized
gut flora which can digest cellulose and use it as a food source. In these animals,
certain species of bacteria reside in the rumen (part of the digestive system of
herbivores) and secrete the enzyme cellulase.
Cellulose-digesting bacteria also live in the appendix of grazing animals. The
appendix is, therefore, important in the digestive systems of ruminants, and this
may explain why it seems to be a vestigial organ in humans.

Dietary Fiber

Dietary fiber refers to mainly indigestible carbohydrates found in plant-based

foods. Most dietary fiber exists in the form of polysaccharides.
Dietary fiber can be divided into two groups:

 Soluble fiber: such as inulin, pectin, or xylose.

 Insoluble fiber: such as cellulose, chitin, resistant starch.

A few starches, known as resistant starches, are classified as dietary fiber.

Dietary fiber is highly beneficial to the digestive system, as it promotes regular
bowel movements by adding bulk to stools in the digestive system, and it slows
down the absorption rate of glucose from food. Fiber also helps remove excess
cholesterol from the body. It binds to cholesterol in the small intestine and trans-
ports cholesterol out of the body through feces.

Proteins

Moving on from carbohydrates, we will next look at proteins and amino acids.
The word protein comes from the Greek word “proteios”, which means first or
primary. Proteins, the building blocks of life, are synthesized in all forms of living
cells. Humans have tens of thousands of unique proteins, which are all constructed
from the set of 20 amino acids.
Multiple amino acids connected by peptide bonds form a polypeptide. How-
ever, it is important to note that the term polypeptide is not synonymous with
32 2 Introduction to Biological Macromolecules

protein. A functional protein does not need to be formed by a single polypep-

tide chain but can actually be made of multiple polypeptides precisely folded into
a unique molecular shape. This specific protein structure determines its final func-
tion.

Amino Acids

As mentioned above, there are 20 different amino acids, and they share common
structures, namely: an amino group and a carboxyl group connected by an alpha
carbon (Fig. 2.9). Amino acids can be linked via peptide bonds between the amino
and carboxyl groups of different amino acids, forming a polypeptide.
Amino acids differ through their R group or side chain, which is also bound
to the central carbon. The chemical properties of this side chain determine the
functional role of the amino acid and modulate the characteristics of polypeptides.
Of the 20 different amino acids, nine are described as essential, meaning they
cannot be made in the cells of our body and must be isolated from our diet.

Peptide Bonds

Amino acids are connected by a dehydration reaction, marked by the removal

of water. The resulting covalent bond is called a peptide bond (amino bond).

Fig. 2.9 Basic structure

of an amino acid. Structure
of a representative amino
acid. All amino acids con-
tain an amino group and
a carboxyl group joined to
a central carbon atom. The
side group varies between
amino acids, and it gives
rise to their unique chemi-
cal and physical properties
2.2 Introduction to Biological Macromolecules Theory Content 33

Fig. 2.10 Peptide bond linkage of amino acids. Amino acids are bound by peptide bonds
to form polypeptides. The linkage occurs between the amino and carboxyl group, releasing
H2 0 in the process

A polypeptide, regardless of length, has a single amino acid end (N-terminus) and
a single carboxyl end (C-terminus) (Fig. 2.10).

Protein Structure

Proteins have at least three structures: primary, secondary, and tertiary, with some
having an additional quaternary structure (Fig. 2.11).
The primary structure of a protein is simply its amino acid sequence in a linear
form. The secondary structure consists of the coils (alpha-helix) and folds (beta-
sheet) that result from the hydrogen bonds which form between repeating con-
stituents of the polypeptide chains. The tertiary structure is the overall shape of the
polypeptide resulting from all the interactions between the side chains of various
34 2 Introduction to Biological Macromolecules
2.2 Introduction to Biological Macromolecules Theory Content 35

Fig. 2.11 Schematic view of the different protein structures. The various structures
formed by amino acids and proteins are given specific terms. The primary structure refers to
the linear amino acid sequence. The secondary structure refers to the formation of regular
substructures such as alpha helices or beta sheets. The tertiary structure describes the 3D
structure of the protein, accounting for the way the substructures interact with each other. If
a protein is comprised of multiple polypeptides, this describes the quaternary structure
J

amino acids. A quaternary structure arises when a protein consists of two or more
polypeptide chains.

Lipids

Lipids are a group of water-insoluble compounds found in the tissue of plants and
animals. Common lipids include:

 Fats
 Phospholipids
 Sphingomyelins
 Waxes
 Sterols

The main functions of lipids are energy storage, mobilization, and utilization.
Other functions of lipids include the synthesis of prostaglandin and cytokines, cell
differentiation and growth, cell membrane structure, signal transmission, hormone
synthesis, and bile acid synthesis.

Fats

A fat molecule, also known as a triglyceride, is constructed from two kinds of

smaller molecules: fatty acids and glycerol.
Fatty acids are the building blocks of fat. To form a fat molecule, three fatty
acid molecules are joined to glycerol by an ester linkage. Ester linkages are formed
during the dehydration reaction that occurs between a hydroxyl and a carboxyl
group. You will be able to observe this molecular process in the Introduction to
(Food) Macromolecules. Based on the type of the fatty acid, fats can be saturated
or unsaturated.
36 2 Introduction to Biological Macromolecules

Fat mostly refers to fats that are solid at room temperature. Fats that are liquid
at room temperature are called oils.

Essential Fatty Acids

As the name suggests, essential fatty acids are fatty acids essential for, but not
synthesized by the human body, for example: omega-6 fatty acids and omega-3
fatty acids. Omega-6 and omega-3 fatty acids are essential for health and can only
be obtained through the diet.
Foods high in omega-3 and omega-6 fatty acids include vegetables, oils, seeds,
nuts, fish, and other seafood.

Saturated Fatty Acids

In saturated fatty acids, all of the carbon atoms are saturated or “filled” by carbon
atoms, meaning no carbon–carbon double bonds exist in their structure.
Most saturated fatty acids are straight hydrocarbon chains and due to this struc-
ture are able to pack closely together, meaning most are solid at room temperature.
Meat and dairy products are typically rich in saturated fats (Fig. 2.12).

Unsaturated Fatty Acids

Unsaturated fatty acids contain double bonds in their structure. The double bond
is in the cis configuration, which means that the hydrogen atoms attached to the
carbon atoms at either end of the double bond are orientated in the same plane,
which causes the fatty acid to kink. Monounsaturated fatty acids contain one dou-
ble bond, whereas polyunsaturated fatty acids have two or more double bonds in
their structure (Fig. 2.12).
Due to the kinks introduced into their structure unsaturated fatty acids often
cannot pack together closely enough to solidify. Therefore, most unsaturated fats
are liquid at room temperature. Common sources of unsaturated fats include veg-
etable oils such as olive oil, canola oil, and sunflower oil.
2.2 Introduction to Biological Macromolecules Theory Content 37

Fig. 2.12 Fatty acid molecules. Saturated fatty acids have a linear structure, every carbon
is saturated with hydrogens. In an unsaturated fatty acid, the (cis) double bonds between
carbons generate a kink or bend in the structure of the fatty acid. A trans fatty acid is the
isomer of an unsaturated fatty acid. The “trans” double bond maintains the linear structure
of the molecule

Trans Fatty Acids

Trans fats are uncommon in nature but are often formed when polyunsaturated
fats are hydrogenated (have hydrogens added to “saturate” the carbon atoms of the
double bond) to improve shelf life and character (by standardizing the fatty acid
length giving a constant melting temperature) by breaking double bonds.
A trans fatty acid is the isomer of an unsaturated fatty acid. The double bond
is in trans configuration, meaning that the hydrogen atoms attached to the carbon
atoms at either end of the double bond are orientated on opposite sides; this trans
fatty acid, therefore, remains straight.
Several studies have identified that trans fats may increase the risk of heart
disease and various cancers, and so if you want to avoid eating trans fats, avoid
consuming products prepared using hydrogenated vegetable oils like margarine.
38 2 Introduction to Biological Macromolecules

The most common foods that contain trans fats are commercially prepared baked
foods (cookies, pies, donuts, etc.), snack foods, and processed foods, including fast
foods (Fig. 2.12).

Nucleic Acids

Nucleic acids include DNA and ribonucleic acid (RNA), which are the most impor-
tant biological macromolecules. While fundamental to life, they are not covered in
the Introduction to Biological Macromolecules simulation, which focuses on food
macromolecules.
Basically, nucleic acids are essential in all organisms to encode and store ge-
netic information, which is then expressed to control cellular functions and trans-
mitted to future generations.
In humans, DNA functions as the long-term storage molecule for genetic in-
formation. The nucleus of every cell contains 23 chromosome pairs comprised
of approximately three billion nucleotides. In contrast, RNA is a more transient
molecule found in the nucleus and cytoplasm and is used in the process of pro-
tein synthesis from the genetic information stored in DNA. Furthermore, RNA
molecules have a variety of regulatory roles. Interestingly, other organisms, such
as viruses, use RNA for both storage and regulatory purposes.
As mentioned previously, DNA as well as RNA polymers are long helical struc-
tures comprised of nucleotides. However, while RNA remains a single strand,
DNA most often exists as two strands wrapped around each other, forming the fa-
mous double-helix structure described by Watson, Crick, Wilkins and Franklin in
their groundbreaking research (Watson and Crick 1953).
A nucleotide monomer contains a 5-carbon sugar, a phosphate group, and a ni-
trogen base. If the 5-carbon sugar is ribose (C5 H10 O5 ), the formed nucleic acid will
be RNA, whereas if the sugar is deoxyribose (C5 H10 O4 ), as the name suggests, the
formed nucleic acid will be DNA.
There are five nitrogenous bases adenine (A), cytosine (C), guanine (G),
thymine (T), and uracil (U), and due to the exact order of the nucleotides, ge-
netic information can be stored in the nucleic acid. Importantly, thymine is found
only in DNA, whereas uracil is found only in RNA.
2.2 Introduction to Biological Macromolecules Theory Content 39

Biochemical Assays

Biochemical assays are often performed to identify or quantify the presence or

absence of certain molecules within a sample. Below, we introduce four simple
tests commonly used to determine the macromolecules discussed above.

Benedict’s Test
This test is used to detect reducing sugars, typically monosaccharides or disac-
charides. Benedict’s solution is a blue colored liquid that contains copper sulfate
(CuSO4 ). In the presence of reducing sugars such as glucose, fructose, lactose,
maltose, or galactose, the copper binds to the oxygen in the free aldehyde or ke-
tone group, forming a copper oxide, turning the solution brown.
It shows a negative result for non-reducing sugars such as sucrose or starch and
remains blue.

Iodine Test
This test is used to detect starch. Iodine solution is normally a pale-yellow color
in the presence of starch, however, the iodine molecule binds with starch, inducing
a color change, with the solution becoming a dark bluish black.
Monosaccharides and other polysaccharides do not cause a color change.

Biuret’s Test
This test is used to detect and quantify peptides in a sample. At rest, Biuret’s so-
lution is a blue liquid that contains copper sulfate (CuSO4 ) and sodium hydroxide
(NaOH). In the presence of peptides, copper sulfate actively binds to the peptide
bonds, forming a structure that becomes a violet color in an alkaline environment.
The sodium hydroxide in the test provides the alkaline environment.

Sudan IV (Red) Test

This test uses Sudan IV as a fat-soluble dye that stains lipids red. Two parameters
are expected in positive results: Firstly, two distinct layers should be visible, indi-
cating the presence of water-insoluble substances. Secondly, the top layer should
appear red, as it has been stained by the Sudan IV dye.
If only a single layer is observed there are no insoluble substances in the sample.
If two layers are observed but the top one is unstained, then the insoluble substance
is not fat.
40 2 Introduction to Biological Macromolecules

2.3 Let’s Get Started

You should now have a solid understanding of biological macromolecules and also
how the science of biochemistry is important for a whole host of processes. By
using your knowledge of macromolecules and armed with information from a va-
riety of biochemical food assays, you will engage your friend in a discussion about
healthy eating.

Techniques Used in the Lab

 Benedict test
 Iodine test
 Sudan test
 Biuret test

Learning Objectives
At the end of this simulation, you will be able to . . .

 Identify the types of macromolecules found in food

 Describe the structure of carbohydrates, proteins and lipids
 Know which test to use to detect macromolecules in food samples

ACCESS THE VIRTUAL LAB SIMULATION HERE www.labster.com/

springer BY USING THE UNIQUE CODE AT THE END OF THE PRINTED
BOOK. IF YOU USE THE E-BOOK YOU CAN PURCHASE ACCESS TO
THE SIMULATIONS THROUGH THE SAME LINK.

Further Reading
Alberts B et al (2015) The molecular biology of the cell, 6th edn. Garland Science, Abingdon
Nelson DL, Cox MM (2013) Lehninger: Lehninger principles of biochemistry, 6th edn. W.H.
Freeman, New York
OpenStax CNX (2018) OpenStax, biology. http://cnx.org/contents/185cbf87-c72e-48f5-
[email protected]. Accessed 1 June 2018
Pratt CW (2011) A biology laboratory exercise using macromolecule assays to distinguish
four types of milk. J Microbiol Biol Educ 12:1
Further Reading 41

Urey LA et al (2014) Campbell biology, 10th edn. Pearson, Boston

Watson JD, CRICK FHC (1953) Molecular structure of nucleic acids: a structure for de-
oxyribose nucleic acid. Nature 171:737–738
Carbohydrates
3

© Labster ApS under license to Springer Verlag GmbH 2019 43

A. Gardner et al., Labster Virtual Lab Experiments: Basic Biochemistry,
https://doi.org/10.1007/978-3-662-58499-6_3
44 3 Carbohydrates

3.1 Carbohydrate Simulation

Carbohydrates are a particularly important type of macromolecule, providing us

with a large proportion of our energy from the food we eat. Most people are fa-
miliar with carbohydrates in the dietary sense, for example, to lose weight, some
individuals adhere to “low-carb” diets. Athletes, in contrast, often “carbohydrate
load” before important competitions to ensure that they will have enough energy
to compete at a high level. In the Carbohydrates simulation, you will analyze dif-
ferent carbohydrates at the molecular level, before recommending a training and
diet plan to your friend.

Different Types of Carbohydrates

Carbohydrates are an essential part of our diet because they provide energy to the
body. Grains, fruits, and vegetables are all natural sources of carbohydrates for
energy. These foods consist of both soluble and insoluble carbohydrates. The
insoluble part is known as fiber, which is mostly cellulose. Understanding how
our body deals with the various types of carbohydrates is key in learning how
we derive the energy we need to function. Use the in-lab molecule visualizer to
study the chemical structure of sugars and learn the basic molecule structures and
chemical formulas (Fig. 3.1).

Learn How Carbohydrates Are Digested

Your goal in the Carbohydrates simulation is to learn how carbohydrates are di-
gested and utilized by the body as an energy source. You will perform an experi-
ment to get a sense of how the amylase breaks down starch, before watching a 3D
animation to visualize the molecular process of carbohydrate digestion, showing
you exactly what occurs inside our bodies after eating (Fig. 3.2).

Test the Effect on Blood Glucose Levels

You will find that the effect of different foods on blood glucose level is very dif-
ferent, depending on the carbohydrates they contain. Choose different food items
and measure the blood glucose level of a virtual test subject to see this in real time.
Will you be able to use your carbohydrates knowledge to figure out which foods
cause a spike in the blood glucose levels (Fig. 3.3), and how an athlete might use
this information?
3.1 Carbohydrate Simulation 45

Fig. 3.1 Observe the chemical structure of various sugar molecules using the molecule vi-
sualizer in the Carbohydrates simulation

Fig. 3.2 The Carbohydrates simulation uses 3D animation to show you how our bodies
digest carbohydrates
46 3 Carbohydrates

Fig. 3.3 Measure blood glucose levels after altering various parameters in the Carbohydrates
simulation

3.2 Carbohydrates Theory Content

You will already have a basic understanding of carbohydrates from the Introduction
to Biological Macromolecules simulation. Here, we dive deeper into carbohy-
drates, exploring how they’re broken down by the digestive system and taken up
into the bloodstream, and what effect this has on our bodies. The theory content
below will equip you with all the knowledge you’ll need to complete the Carbohy-
drates simulation successfully.

Carbohydrates

As you know, macromolecules are built from monomers. In the case of carbohy-
drates, they are built from single-sugar monomers. They can exist as simple, sin-
gle-sugar molecules (monosaccharides), or chains of two or more sugar molecules
(disaccharides and polysaccharides). Carbohydrates are an important source of
energy and structural material for organisms.
Carbohydrates are composed of carbon (C), hydrogen (H), and oxygen (O)
atoms, typically in a 1 : 2 : 1 ratio, which can be represented by the stoichiomet-
3.2 Carbohydrates Theory Content 47

ric formula Cm (H2 O)n (where m and n can differ). For example, glucose has the
formula C6 H12 O6 , which would be represented as C6 (H2 O)6 . This formula also
explains the origin of the term “carbohydrate”, which means watered (“hydrate”)
carbon (“carbo”).
As discussed previously, carbohydrates are an essential part of a diet because
they provide energy to the body. Grains, fruits, and vegetables are all natural
sources of carbohydrates that can be used for energy. These foods consist of both
soluble and insoluble carbohydrates; the insoluble part is known as fiber, which is
mostly cellulose. For more general information, see the carbohydrates section of
the Introduction to Biological Macromolecules chapter of this book.

The Digestive System

The digestive system is designed to facilitate the transformation of food matter

into its nutrient components. The mouth, esophagus, stomach, small and large
intestines, and liver are part of the digestive tract through which food passes. In
addition, the accessory organs, the gallbladder and pancreas, add secretions (en-
zymes) and are regulated by hormones in response to the food consumed (Fig. 3.4).

The process of digestion is as follows:

Mouth The mouth is the point where food enters into the digestive system. Here,
food is broken into smaller particles by the chewing action of the teeth. The process
of digestion actually begins in the mouth, with saliva, produced by the salivary
glands and containing the enzyme amylase, mixing with the food, catalyzing the
hydrolysis of starches into sugars. Another enzyme called lipase is produced by
the cells in the tongue. Whereas amylase begins the breakdown of starch, lipase
begins the breakdown of fat components in the food. The chewing and wetting
action provided by the teeth and saliva prepare the food into a mass called the
bolus, ready for swallowing.

Esophagus The esophagus is the tubular organ connecting the mouth and stom-
ach. The bolus passes through the esophagus after being swallowed. Under the
control of smooth muscles which line the esophagus and undergo a series of wave-
like movements called peristalsis, the bolus is pushed towards the stomach. This
peristaltic wave is unidirectional, meaning that it moves the bolus from the mouth
to the stomach, but does not allow movement in the other direction. This peristaltic
48 3 Carbohydrates
3.2 Carbohydrates Theory Content 49

Fig. 3.4 Schematic of the human digestive system. The functions of the various organs of
the digestive system are explained in the section below. Briefly, food enters the body through
the mouth (A), before being swallowed into the esophagus (B) for transport to the stomach
(C). Here, the process of breaking down food into its components begins, with digested food
then passing into the small intestine (D) for absorption for further processing. Undigested
food and waste products then pass into the large intestine (E) for excretion. The liver (F)
gallbladder (G), and pancreas (H) all secrete key enzymes and chemicals required to digest
food properly
J

movement is an involuntary reflex which takes place in response to the bolus en-
tering the esophagus.

Stomach A large portion of digestion occurs in the stomach, a sack-like organ

that secretes gastric digestive juices and maintains an acidic environment to aid
digestion. The pH in the stomach is between 1.5 and 2.5, and this highly acidic
environment is required for the efficient chemical breakdown of food and the ex-
traction of nutrients. When empty, the stomach is a rather small organ; however, it
can expand to up to 20 times its resting size when filled with food.
Proteins are broken down in the stomach by the enzyme pepsin. Pepsin is se-
creted in an inactive form called pepsinogen. Pepsin breaks peptide bonds and
cleaves proteins into smaller polypeptides. It also helps to activate more pepsino-
gen, beginning a positive feedback mechanism. Moreover, parietal cells in the
stomach secrete hydrogen and chloride ions, which combine in the lumen (the open
region) of the stomach to form hydrochloric acid (HCl). HCl helps to convert the
inactive pepsinogen to pepsin, and the highly acidic environment also kills most
microorganisms present in the food (thus preventing infection). HCl in combina-
tion with the activity of pepsin results in the hydrolysis of proteins in the ingested
food. This chemical digestion is aided by the churning action of the stomach, en-
suring the stomach contents are well mixed for optimal processing. The stomach
empties itself within 2 to 6 h after a meal, but only a small amount of the food
(gastric juice mix), known as chyme, is released into the small intestine at a time.

Small intestine The digestion of protein, fats, and carbohydrates is completed

in the long tube-like small intestine. The intestinal epithelium is folded many
times, forming structures known as villi, with each individual cell also featuring
numerous folds on its surface (microvilli). These all act to increase the surface area
of the intestine and increase the efficiency of nutrient absorption into small blood
vessels surrounding the intestine. These small vessels join up, forming the hepatic
50 3 Carbohydrates

portal vein carrying the nutrients to the liver. The small intestine is surrounded
by two layers of smooth muscles that contract in a wavelike pattern similar to the
esophagus. This peristaltic movement mixes the contents of the small intestine and
slowly moves it towards the large intestine.

Large intestine The large intestine reabsorbs water from the undigested food ma-
terial and processes waste material. The human large intestine is much smaller in
length compared to the small intestine but larger in diameter. The above organs
are in direct contact with the food we ingest, however, they require the assistance
of accessory organs including the liver, gallbladder, and pancreas to digest food
efficiently.

Liver The liver carries out important roles such as the digestion of fats and the
detoxification of the blood. The liver also produces bile, a digestive juice that is
required for the breakdown of the fatty components within food in the first part
of the small intestine. The liver also processes vitamins and fats and synthesizes
many plasma proteins for distribution around the body.

Gallbladder The gallbladder is a small organ that aids the liver by storing bile
and concentrating bile salts. When chyme containing fatty acids enters the small
intestine, the bile is secreted from the gallbladder to aid digestion.

Pancreas The pancreas is an important gland in the animal digestive system that
secretes digestive juices. The chyme produced from the stomach is highly acidic
in nature. The pancreatic juices contain high levels of bicarbonate, an alkali that
neutralizes the acidic chyme. Additionally, the pancreatic juices contain a large
variety of enzymes that catabolize starches, disaccharides, proteins, and fats for
easier absorption in the small intestine.

Glycemic Index

The glycemic index (GI) represents the total rise in a person’s blood sugar level
after consumption of a specific food.
The GI is measured following a 12-hour fasting period and ingestion of a food
with a fixed amount of available carbohydrate (usually 50 g). It is measured from
the incremental area under the 2-hour blood glucose response curve. As the GI
of food is calculated relative to the equivalent of 50 g glucose, it typically ranges
between 50 and 100. For example, the GI of pure glucose is defined as 100.
3.2 Carbohydrates Theory Content 51

While GI is useful for understanding how the body responds to various carbohy-
drate intakes, it only accounts for available carbohydrates (i.e., total carbohydrates
excluding fibers) in a particular food. Furthermore, an increase in blood sugar can
also be influenced by a number of other factors, such as the quantity of fat eaten
with the food.
The GI does not take the quantity of food into account, but only the carbo-
hydrates contained within. A related measure, the glycemic load, factors in the
quantity of food by multiplying the carbohydrate content of the serving with the
GI of the food of interest. For example, carrots have a high GI, but a low glycemic
load for the quantity typically consumed. In contrast fructose, has a low GI, but
can have a high glycemic load, if a large quantity is consumed.

Blood Glucose Regulation

In order to manage nutrient intake, the body uses hormones to moderate energy
stores. Pancreatic cells produce the two hormones insulin and glucagon to regulate
the blood glucose level. These two hormones maintain a homeostatic glucose level
(Fig. 3.5).

Insulin

Insulin is a peptide hormone produced by the beta cells of the pancreas and func-
tions to increase the rate of glucose uptake and utilization in targeted cells. As
such, insulin lowers blood glucose levels, with circulating glucose moving into the
cells. Impaired insulin function can lead to a condition called diabetes mellitus.

Insulin and Blood Glucose Level

When the blood glucose level rises (for example, after a meal is consumed), insulin
is released into the bloodstream to lower the level by promoting cellular uptake. It
also stimulates the liver to convert glucose to glycogen, which is then stored in
cells for later use.
This happens via an insulin-mediated increase in the number of glucose trans-
porter proteins in cell membranes, which removes glucose from circulation by
facilitated diffusion. As insulin binds to its target cell via insulin receptors, it trig-
gers a signaling cascade, which ultimately triggers the cell to incorporate glucose
52 3 Carbohydrates

Fig. 3.5 Regulation of blood glucose. Two enzymes, insulin (yellow) and glucagon (blue)
are secreted by the pancreas to regulate blood sugar level. Insulin is secreted when blood
sugar is high (yellow pathway) promoting the uptake of glucose from the blood into tissues
and also the conversion of glucose to glycogen in the liver. When the blood sugar level
drops, glucagon is secreted (blue pathway), which promotes the conversion of glycogen into
glucose in the liver, thus raising the blood sugar level
3.2 Carbohydrates Theory Content 53

transport proteins into its membrane. These transport proteins move glucose into
the cell, where it can be used to generate ATP. Some tissues, such as the brain and
the liver, do not require these transporters and can freely absorb glucose without
insulin.
Insulin also stimulates adipocytes to convert glucose into fat as a storage mech-
anism and the synthesis of proteins. These actions mediated by insulin cause
a decrease in the blood glucose concentration, a hypoglycemic or “low sugar”
effect, which inhibits further insulin release from beta cells through a negative
feedback loop.

Glucagon and Blood Glucose Level

The other major hormone controlling blood glucose level is glucagon. When blood
glucose levels decrease, for example during exercise or between meals, glucagon
is secreted by alpha cells in the pancreas. Glucagon promotes the breakdown of
glycogen stored in the liver and muscle cells back into glucose via a process known
as glycogenolysis. This released glucose can then be used as an energy source in
the muscle cells and released into the circulatory system from the liver, which can
then be used by other cells.
At the same time, glucagon also promotes the absorption of amino acids from
the blood by the liver, where they are then converted to glucose via a process known
as gluconeogenesis. Finally, glucagon also stimulates adipose cells to release fatty
acids into the blood, which can be utilized as an alternative energy source of the
body.
Together these actions result in an increase in blood glucose levels, and this
increase inhibits further production of glucagon in the pancreas, forming a negative
feedback loop.

Diabetes Mellitus

As mentioned above, glucose is an important energy source for the human body. In
order to use that energy, the body requires the hormone insulin, which stimulates
cells to uptake and use glucose. In diabetes mellitus, commonly referred to as
diabetes, not enough insulin is produced or it doesn’t function correctly. This
results in a high level of glucose in the blood and a relatively lower level in the cells
where it is actually needed. Two types of diabetes have been described: type 1 and
type 2.
54 3 Carbohydrates

Type 1 diabetes occurs when the body doesn’t produce enough insulin to regu-
late blood glucose. It is typically diagnosed early in life and is not modulated by
diet or age. Conversely, type 2 diabetes often develops later in life and is associated
with a variety of lifestyle and genetic risks, with diet, obesity, and lack of exercise
being the major causes. It arises due to the development of insulin resistance in the
liver. In health, insulin suppresses the release of glucose from the liver; however,
in those with type 2 diabetes, this activity is lost, and the liver continues to secrete
glucose into the blood. This is also associated with a lack of insulin production
generating a vicious negative cycle.

3.3 Let’s Get Started

So now you’ve built on your early knowledge of macromolecules and focused in

on carbohydrates. You now know how important they are to life, providing us the
energy to breathe, think, and move, and lots of the building blocks our cells need.
Will you be able to apply your carbohydrate knowledge to help your friend fine-
tune their diet in the Carbohydrates simulation?

Techniques Used in the Lab

 Reading and interpreting scientific papers
 Analyzing blood sugar measurements

Learning Objectives
At the end of this simulation, you will be able to . . .

 Describe the molecular structure of sugars and polysaccharides

 Understand digestion and appreciate the complexity of the human body
 Experiment with different foods and measure their impact on the blood
sugar level

ACCESS THE VIRTUAL LAB SIMULATION HERE www.labster.com/

springer BY USING THE UNIQUE CODE AT THE END OF THE PRINTED
BOOK. IF YOU USE THE E-BOOK YOU CAN PURCHASE ACCESS TO
THE SIMULATIONS THROUGH THE SAME LINK.
Further Reading 55

Further Reading
Alberts B et al (2015) The molecular biology of the cell, 6th edn. Garland Science, Abingdon
Nelson DL, Cox MM (2013) Lehninger: Lehninger principles of biochemistry, 6th edn. W.H.
Freeman, New York
OpenStax CNX (2018) OpenStax, biology. http://cnx.org/contents/185cbf87-c72e-48f5-
[email protected]. Accessed 1 June 2018
Urey LA et al (2014) Campbell biology, 10th edn. Pearson, Boston
Enzyme Kinetics
4

© Labster ApS under license to Springer Verlag GmbH 2019 57

A. Gardner et al., Labster Virtual Lab Experiments: Basic Biochemistry,
https://doi.org/10.1007/978-3-662-58499-6_4
58 4 Enzyme Kinetics

4.1 Enzyme Kinetics Simulation

In the Enzyme Kinetics simulation, you will see how catalysis increases the rate of
chemical reactions, with a focus on the role of enzymes in this process. You will
learn all about the kinetics of enzymes with the Michaelis–Menten equation and re-
action rate constants through an example linking DNA mutations with enzyme hy-
peractivity. Finally, you will run experiments using the wild-type and the mutated
enzyme alcohol dehydrogenase in the context of learning about alcohol flush syn-
drome and play with inhibitors that alter the enzymatic reaction in various ways.

Use a Spectrophotometer to Measure Enzyme Activity

In the Enzyme Kinetics simulation, you will have access to a fully equipped work-
bench where you can prepare the alcohol dehydrogenase reaction and measure the
production of acetaldehyde using a spectrophotometer (Fig. 4.1). You will learn
about the concept of spectrophotometry, how to prepare a master mix and how to
calculate dilutions to ensure experimental accuracy.

Observe How Enzymes Work at the Molecular Level

Detailed interactive 3D animations illustrate what happens at the molecular level
when the substrate and cofactor enter the active site of an enzyme (Fig. 4.2).

Fig. 4.1 Use a spectrophotometer to measure enzyme activity in the Enzyme Kinetics sim-
ulation
4.1 Enzyme Kinetics Simulation 59

Fig. 4.2 Observe the molecular interactions between enzymes, substrates and co-factors in
the Enzymes Kinetics simulation

Fig. 4.3 Experiment with enzymes freely in the Enzyme Kinetics simulation in order to
better understand alcohol flush syndrome
60 4 Enzyme Kinetics

Throughout the simulation you will be able to test your understanding of the key
concepts and to reinforce your learning.

Experiment Freely and Measure the Results

For every measurement, you will generate a progress curve displaying the amounts
of product formed over time. By analyzing the data and plotting your own
Michaelis–Menten graph you will be able to find two key measurements of en-
zyme activity: the K m and V max for each enzyme. By comparing K m and V max
values of the wild-type versus mutant alcohol dehydrogenase, you will be able to
understand the mechanisms underlying alcohol flush syndrome (Fig. 4.3).

4.2 Enzyme Kinetics Theory Content

The theory content below contains all the details of the important formulae re-
quired to measure the activity of the enzyme alcohol dehydrogenase (ADH) and
also the practical details of enzyme kinetics experimentation, required to complete
the Enzyme Kinetics simulation successfully.

Enzymes

Enzymes are proteins that act as catalysts for specific reactions. By providing
an alternative reaction requiring lower activation energy, they allow the reaction
to proceed at a much higher rate. It is important to note that enzymes do not
change the equilibria of a reaction, they only increase the reaction rate. Without the
enzyme, the reaction would, therefore, still proceed in the same direction; however,
it would be slower, often a lot slower. As a proper catalyst, the enzyme itself will
return to its initial state after the reaction and is not consumed in the process.
A single-enzyme molecule can, therefore, catalyze thousands of reactions before
being degraded. This can be represented by the formula below:

At equilibrium the forward and reverse rate will be balanced, and there will be
no net change in the concentrations of either the reactants (A C B) or the products
4.2 Enzyme Kinetics Theory Content 61

(C C D). An enzyme does not change this equilibrium but instead increases the
forward reaction rate.
Enzymes are required by our body to perform specific metabolic reactions.
They are often highly specific for their substrates, much like a key is specific to
a lock. The specificity of enzymes for their substrates led Emil Fischer to pro-
pose the so-called “lock and key” hypothesis in 1894. However, the “lock and
key” hypothesis implies that enzymes are static molecules, which they are not.
Another mechanism, called “induced fit” is the preferred model and according to
this model, the enzyme undergoes conformational changes when binding to the
substrate, leading to the activation of the catalytic function.

Enzymes and Activation Energy

There are many different ways by which enzymes enhance the rate of their spe-
cific reactions. When two substrates are converted into one or more products, the
enzyme will bind both substrates, thereby ensuring that they are in close vicinity
and correctly oriented towards each other. When the reaction occurs without the
enzyme, the two substrates have to randomly bind with each other from the correct
angle and with enough energy (speed) to overcome the much higher activation en-
ergy, which makes this process a much more unlikely event. When the substrates
are bound by an enzyme, these challenges are overcome due to the structure of the
enzyme (Fig. 4.4).
It is important to note that thermodynamically the enzyme does not provide
additional free energy to reach the transition state of the reaction, but instead offers
a different chemical pathway with a lower amount of activation energy needed for
a transition state to occur.
The activation energy and the reaction rate are directly linked through the Ar-
rhenius equation:
Ea
k D Ae RT
where k is the reaction rate, A the pre-exponential factor, Ea the activation energy,
T the temperature in Kelvin, and R the gas constant.
Therefore, the enzyme lowering the required activation energy has a direct im-
pact on the reaction rate, which can be measured.
62 4 Enzyme Kinetics

Fig. 4.4 Energy requirements for reactions. A reaction from a substrate to product is
a transition from one energy state to another. A transition state exists between the substrate
and product. This state has a higher energy level than both the substrate and product. A cat-
alyst, such as an enzyme, will provide a different transition state with a lower energy level,
so that the transition energy is reached more easily, and this results in a faster reaction

Substrates

Substrate refers to the initial molecules converted into a product during a reaction.
If we think of the enzyme as a machine in an assembly line, the substrate would
be the raw materials, and the products would be the finished items. Similarly, the
substrate is consumed in the process, leading to a decrease in its concentration in
the system.
Throughout this simulation, we use ADH as an example. The substrate for ADH
is ethanol; however, ADH can also bind other substrates with a similar structure
such as methanol. In the same way that ethanol is converted to acetaldehyde,
4.2 Enzyme Kinetics Theory Content 63

methanol is converted to formaldehyde by ADH. Thus, ADH is an example of

a less specific enzyme with an affinity for several substrates. Some enzymes are
very specific and will only catalyze one reaction, whereas others, such as ADH,
are more flexible.

Cofactors

Some enzymes require external help, often additional molecules for catalysis to
take place (Fig. 4.5). These helper molecules are called cofactors. Cofactors are
non-protein molecules that bind to the enzyme and contribute to reactions in a num-
ber of different ways, for example promoting binding of the substrate, or altering
the shape of the enzyme. Cofactors can either be inorganic ions, such as the Zn2+
ions required by ADH, or they can be more complex organic or metallo-organic
molecules, which are then called coenzymes. If a cofactor is bound tightly (some-
times covalently) to the enzyme, it is termed a prosthetic group.

Products

The products are formed during the reaction of substrates. When the reaction oc-
curs, the concentration of the substrate decreases, while the concentration of the
product increases.
For the reaction catalyzed by ADH, the product is acetaldehyde. ADH oxidizes
ethanol into acetaldehyde by removing two H+ ions and two electrons from the
ethanol substrate.
Most people think that ethanol is the only compound responsible for the feel-
ing of being drunk. However, it has been indicated that many effects associated
with alcohol consumption may be caused by increased levels of acetaldehyde. The
accumulation of acetaldehyde is also the cause of the symptoms of alcohol flush
syndrome (described below).

The Active Site

The area where substrates and cofactors bind to the enzyme is called the active site
(Fig. 4.5). This is where the catalysis of the reaction takes place. An active site
often appears like a pocket and consists of several amino acids, which form tem-
porary bonds with the substrate. However, since a protein is a flexible molecule,
64 4 Enzyme Kinetics

Fig. 4.5 The role of cofactors. Here a cofactor (beige) binds to the enzyme (blue), thereby
contributes to the reaction, and leaves the enzyme with the product in a changed state. This
could, for example, be a changed oxidation state, which then needs to be restored elsewhere
before the cofactor can assist in another reaction. The active site of the enzyme is marked in
red
4.2 Enzyme Kinetics Theory Content 65

the active site can sometimes be formed only in the presence of the substrate or at
a specific temperature or pH, or in the presence of specific cofactors, and might be
difficult to detect in other situations.
While the size the active site itself might look small in comparison of the size
of the enzyme, the remaining amino-acids not involved in the active site are often
vital in providing a structural backbone for the active site, allowing it to form
a functional configuration (marked in red in Fig. 4.5).
If a mutation causes an amino acid substitution on the residues involved in the
active site, the kinetic parameters may be critically altered, as is the case with
alcohol flush syndrome.

Alcohol Dehydrogenase

ADH catalyzes the oxidation of a broad range of substrates containing hydroxyl

groups, including ethanol. In this case, ethanol is converted into acetaldehyde,
another toxic compound, which is then further metabolized and degraded.
To proceed, the reaction requires a cofactor; here the oxidizing agent nicoti-
namide adenine dinucleotide (NAD+ ). NAD+ is a coenzyme that acts as an electron
acceptor, accepting two electrons and an H+ from ethanol. Thus, ADH catalyzes
the following reaction:

CH3 CH2 OH C NADC • CH3 CHO C NADH C HC

Because ADH triggers the faster oxidation of ethanol, it plays a central role
in breaking down commercial alcohol after ingestion, and mutations in ADH can
severely impair the ability to tolerate alcohol.

Alcohol Flush Syndrome

Humans have several different versions (called isozymes) of ADH. Two of

these, called ADH1B*1 and ADH1B*2, differ in only one amino acid residue,
which however results in significant differences in kinetic properties. Where
ADH1B*1 has an arginine residue at position 47, ADH1B*2 has a histidine
residue. ADH1B*2 is more commonly found in East Asian populations, while
ADH1B*1 is more common in Caucasian populations.
The kinetic differences are due to the chemical properties of arginine and histi-
dine. Arginine in ADH1B*1 forms hydrogen bonds with the pyrophosphate group
66 4 Enzyme Kinetics

of NAD+ , however the histidine residue in ADH1B*2 is not able to form as many
bonds due to the different structure of the side chain of the amino acid. This means
that ADH1B*2 does not bind NAD+ as tightly as ADH1B*1. The rate-determining
step of the overall reaction is the dissociation of NADH. Therefore, the efficiency
of ADH1B*2 is higher, because NADH is not bound as tightly and is thus released
more quickly. Furthermore, because the pK a value of histidine is lower than that
of arginine, the optimal pH of ADH1B*2 (8.5) is lower than that of ADH1B*1
(10.0).
Individuals possessing the ADH1B*2 isozyme experience a condition called
alcohol flush syndrome. The condition leads to flushing of the skin and other
symptoms usually associated with hangovers after the consumption of even small
amounts of alcohol. These symptoms are caused by an elevated level of acetalde-
hyde in the blood, which is due to the higher activity of ADH1B*2 compared to
ADH1B*1. Therefore, the single-amino acid substitution in ADH1B*2, caused by
a mutation in its DNA sequence, leads to alcohol flush syndrome.

Enzyme Kinetic Assays

Enzyme kinetics is the study of enzyme mechanisms through the determination

of reaction rates under varied conditions. The rate of a reaction is dependent on
several factors, including the concentration of the substrate and the enzyme, tem-
perature, pH, and the presence of inhibitors. With careful planning, kinetic assays
can reveal the reaction rates of the different reaction steps, the maximum efficiency
of an enzyme under different conditions, and even the mode of the action of en-
zyme inhibitors.

Performing Kinetic Assays

The aim of a kinetic assay is to model the reaction rate V as a function of the
substrate concentration [S]. It is, therefore, necessary to measure the rate at dif-
ferent initial substrate concentrations. For each substrate concentration, a progress
curve showing the amount of product formed as a function of time is obtained. The
rate of the reaction is approximately constant early in the reaction, however, as the
substrate is used up, the rate decreases, and the progress curve reaches a plateau
when all the substrate has been turned into product (Fig. 4.6). Because [S] changes
during the reaction, it is common to measure the initial reaction rates (V 0 ) while
[S] is high and, therefore, does not influence the reaction and plot these against the
4.2 Enzyme Kinetics Theory Content 67

Fig. 4.6 Measurements of enzyme kinetics. The concentration of product increases over
time, as the enzyme converts the substrate. When the substrate starts to run out in the sys-
tem, less product is formed, leading to a plateau in enzyme activity. To limit the impact
of diminishing substrate concentration on the measurement of enzymatic activity, only the
initial slope V 0 is taken into account

starting substrate concentrations; V 0 is measured from the slope of the progress

curve at the beginning of the reaction, when the progress curve is still linear.
The simplest model of V as a function of [S] is the Michaelis–Menten equa-
tion (see below). Reactions where the Michaelis–Menten equation can be applied
show an increase in the reaction rate when [S] is increased; the more substrate
is available, the easier it is for the reaction to proceed. However, this increase is
diminished as the rate approaches the maximum velocity, or V max . Once V max is
reached, adding more substrate does not affect the reaction rate, as the enzyme is
already saturated (i.e., there is no pause between the consecutive conversions of
two substrate molecules).

Factors Affecting Enzyme Kinetics

Temperature and pH can also affect the reaction rate. Enzymes have an optimum
pH, which is dependent on the composition of the enzyme. This is due to the
properties of the amino acid side chains which comprise the enzyme; some side
chains need to be protonated or deprotonated in order to make a functional en-
zyme. Whether an amino acid side chain is protonated or deprotonated depends
on the pK a of the side chain and the pH of the solution. For instance, histidine has
a pK a of 6.0, which means that it will be mostly protonated at pH < 6.0 and mostly
68 4 Enzyme Kinetics

deprotonated at pH > 6.0. Note that the pK a of side chains may be altered by the
environment, and therefore the pK a of the side chains in enzymes is usually not the
same as those of free amino acids. Temperature also affects the reaction rate. An
increase in temperature leads to an increased reaction rate; however, at a certain
temperature, depending on the enzyme, the enzyme will start to denature. At this
point, the reaction rate will start to decrease and eventually drop to zero when no
enzyme is left structurally able to perform the reaction.

Michaelis–Menten

The Michaelis–Menten model is a simple model of an enzymatic reaction devel-

oped by Leonor Michaelis and Maud Menten in 1913. The model is based on the
following two assumptions:

 An enzymatic reaction proceeds in two steps: formation of an enzyme-substrate

complex (ES) and dissociation of the enzyme (E) and the product (P).
 After a (very) short period of time, the concentration of the ES complex reaches
a steady state, where the rate of formation of ES equals the rate of its consump-
tion.

The first assumption implies that the enzymatic reaction is made up of four
different reactions: formation of ES from E and S, dissociation of ES into E and S
or dissociation of ES into E and P, and the formation of ES from E and P (i.e., the
reverse reaction). The rate of a reaction is usually measured at the beginning of
the reaction, where no significant amount of P has been formed, and therefore the
formation rate of ES from E and P can be ignored. This results in the following
overall reaction.
k1 k2
E C S • ES ! E C P
k1

With k1 , k1 , and k2 the respective reaction rates of each reaction.

This reaction implies that the rate of formation of products, i.e., the reaction
rate, is given by V D k2  ŒES. When almost all the enzyme is part of the enzyme-
substrate complex, the reaction approaches its maximum velocity (V max ). In the
above reaction, assuming there is far more substrate than enzyme, k2 is the rate-
limiting step, and we can consider [ES]  [E], since all enzymes will be in a com-
plex with the substrate, which is in saturating concentration. V max can, therefore,
be expressed as k2  [E].
4.2 Enzyme Kinetics Theory Content 69

The rate-limiting rate constant is also called kcat or the turnover number, and in
the above reaction, kcat D k2 . This means that Vmax D kcat  ŒE.
The above assumptions and definitions give us the important Michaelis–Menten
equation:
Vmax  ŒS
V0 D
Km C ŒS
Where Km D .k1 C k2 /=k1 is the Michaelis–Menten constant, corresponding
to half of the reaction rate V max and where the “0” in V 0 implies that this equation
is only valid for describing the initial rates, where no significant amount of product
has been formed (Fig. 4.7). The two constants V max and K m will be described in
more detail in later sections.

Fig. 4.7 Michaelis–Menten curve fitted to various initial rates V 0 of an enzyme reac-
tion. At low substrate concentrations, the curve is steep, however, at higher concentrations,
the curve reaches a plateau, and the rate approaches V max . The interpretation of K m is also
clear from the figure. K m is equal to the substrate concentration where the reaction rate is
1=2  Vmax
70 4 Enzyme Kinetics

Reaction Rate

The Michaelis–Menten equations, as you have seen in the previous section, de-
scribe the rate of a one-substrate enzyme-catalyzed reaction. The parameters V max
and K m can be obtained experimentally for any given enzyme; however, on their
own they provide very little information about the reaction mechanism, such as the
number of discrete steps and their individual reaction rates. The reaction rate (V)
is defined as the rate of formation of products (P) or as the rate of consumption of
reactants (the substrate, S) over a period of time (t). In a first-order reaction, where
one molecule of substrate is converted to one molecule of product, we can write:

dŒP dŒS
V D D
dt dt

Note that the rate of consumption of the substrate is equal to the negative
change in the concentration of the substrate over time, if the reaction is a 1 : 1 sub-
strate/product ratio (one molecule of substrate will be converted into one molecule
of product). The Michaelis–Menten equation described previously is based on the
following reaction mechanism:

k1 k2
E C S • ES ! E C P
k1

This mechanism includes three individual reactions with three different rate
constants:

 E C S ! ES, formation of the enzyme-substrate complex, with the rate constant

k1
 ES ! E C S, dissociation of the enzyme and the substrate, with the rate constant
k1
 ES ! E C P, dissociation of the enzyme and the product, with the rate constant
k2

In the Michaelis–Menten model it is assumed that the third reaction is the rate-
limiting step, and the associated rate constant k2 is also called the turnover number
or kcat . For another reaction mechanism, the turnover number would be defined
differently, for instance, in the following reaction:

k1 k2 k3
E C S • ES • EP • E C P
k1 k2
4.2 Enzyme Kinetics Theory Content 71

Where the last step is the rate-limiting step. Here, kcat is equal to the rate con-
stant of this step, i.e., kcat D k3 . For reactions with more complicated reaction
mechanisms, kcat can be a function of several rate constants.

Km

The Michaelis constant (K m ) is a parameter in the Michaelis–Menten equation.

K m is equal to the substrate concentration where the corresponding reaction rate
is 1=2  Vmax . An enzyme with a low K m , therefore, achieves its half-maximal
velocity at a low substrate concentration, while an enzyme with a high K m needs
high substrate concentrations to achieve this velocity. It has been experimentally
shown that the K m of an enzyme is usually close to the cellular concentration of
its substrate. For an enzymatic reaction involving two steps, where the second
step is rate limiting, K m is approximately equal to the dissociation constant of
the ES complex. In this case, a low K m implies a high affinity for the substrate.
Importantly, this interpretation of K m is only valid for a few enzymes.

The Lineweaver–Burk Equation

Several methods for determining K m exist. The most direct method is to plot
the initial reaction rate V 0 against the initial substrate concentration [S] and use
curve fitting software to fit the Michaelis–Menten equation directly. However, cer-
tain transformations allow determination of K m via linear regression, and these
transformations are also useful when analyzing enzyme inhibition. Several trans-
formations are possible; a simple one is obtained by taking the reciprocal on both
sides of the Michaelis–Menten equation. This leads to the following expression,
called the Lineweaver–Burk equation:

1 1 Km 1
D C 
V0 Vmax Vmax ŒS

This equation shows that a plot of 1=V0 against 1=ŒS should give a plot that
can be fitted by a straight line with the y-intercept 1=Vmax and slope Km =Vmax
(Fig. 4.8). Thus, both V max and K m can be obtained using linear regression. Below
is an illustration of how to interpret the slope and intersects on a Lineweaver–Burk
plot.
72 4 Enzyme Kinetics

Fig. 4.8 Example Lineweaver–Burk equation fitted to a double-reciprocal transfor-

mation of an enzyme kinetic dataset. A double-reciprocal plot will have the y-intercept
1=Vmax ; V max can therefore be obtained by taking the reciprocal to this intercept

V max

In accordance with the Michaelis–Menten equation, the initial reaction rate (V 0 )

increases with increased substrate concentrations [S]. The reaction rate increases
more at lower substrate concentrations, and it eventually reaches a plateau, ap-
proaching the maximum velocity or V max . The maximum initial velocity is reached
when the enzyme is saturated, i.e., when enough substrate is present to ensure that
practically every enzyme is part of an enzyme-substrate complex. Because the
enzyme can never be completely saturated, V max is never fully reached. V max is
dependent on two things: the turnover number of the enzyme (kcat ), and the con-
centration of the enzyme [E].

Vmax D kcat  ŒE

Thus, a higher [E] leads to a higher V max . The more enzymes you use, the more
substrates are converted into products in the same period of time.
4.2 Enzyme Kinetics Theory Content 73

Determining V max

V max can be determined using the Lineweaver–Burk equation as shown in Fig. 4.8:

1 1 Km 1
D C 
V0 Vmax Vmax ŒS

Based on this equation, a straight line fitted to a double-reciprocal plot will have
the y-intercept 1=Vmax and V max can, therefore, be obtained by taking the reciprocal
to this intercept.
With the slope of the line being Km =Vmax, once we know V max, we can now
calculate K m .

K cat

V max is not the best way to characterize enzyme activity because it is dependent on
the enzyme concentration. The catalytic constant (kcat ) or turnover number, is the
number of enzymatic reactions a single saturated enzyme molecule can catalyze
per unit of time, usually expressed in seconds. kcat is, therefore, a better parame-
ter than V max for comparing different enzymes because it measures the enzymatic
activity per molecule of enzyme, independently of the enzyme concentration.
Because kcat is the maximum number of chemical reactions a single enzyme
molecule can catalyze, the maximum velocity (V max ) at a specific enzyme concen-
tration is obtained by multiplying kcat with the concentration of the enzyme [E],
i.e., Vmax D kcat  ŒE. This means that if V max , which can be experimentally mea-
sured, and [E], which is an initial parameter of the experiment, are known, kcat can
be calculated as follows:
Vmax
kcat D
ŒE

Enzyme Inhibition

Enzyme inhibitors are molecules that decrease the activity of enzymes, and knowl-
edge about inhibitors can, for example, be used in developing drugs or in the
study of biochemical pathways, because inhibitors provide a way to interfere with
these pathways. Enzyme inhibitors can be either irreversible or reversible. Ir-
reversible inhibitors decrease enzymatic activity by destroying or blocking the
enzyme through various mechanisms, while reversible inhibitors keep the enzyme
74 4 Enzyme Kinetics

functional and just temporarily block its action. The inhibitors we will study in the
Enzyme Kinetics simulation are reversible inhibitors.

Types of Enzyme Inhibition

The mechanisms of enzyme inhibitors can be classified into three major groups:
competitive inhibitors, uncompetitive inhibitors, and mixed inhibitors. Competi-
tive inhibitors work by binding to the active site of the enzyme in competition with
the substrate. Uncompetitive inhibitors bind to the enzyme-substrate complex at
a site distinct from the active site, but they cannot bind to the enzyme alone, and
mixed inhibitors can bind to both the enzyme and the enzyme-substrate complex
at a site distinct from the active site.
The mechanisms of enzyme inhibition can be thought of as an extension to the
Michaelis–Menten mechanism, and competitive and uncompetitive inhibition can
be regarded as a special case of mixed inhibition:

Where K I and KI0 are the dissociation constants of the enzyme and inhibitor (EI)
and enzyme, substrate and inhibitor (ESI) complex, respectively. Using the same
approach used for deriving the Michaelis–Menten equation, the following equa-
tion for mixed inhibition can be obtained (for the sake of brevity, the intermediate
calculations are not shown):

Vmax  ŒS
V0 D
Km  ˛ŒS  ˛ 0
4.2 Enzyme Kinetics Theory Content 75

Fig. 4.9 Effect of inhi-

bition on the Michaelis–
Menten plot. Assays with
a competitive inhibitor will
have the same V max as the
control without inhibitors
but a different K m , while
uncompetitive inhibitors
would induce a lower V max
but retain similar K m

Where
ŒI
˛ D1C
KI
And
ŒI
˛0 D 1 C
KI0
As you can see, without inhibitors (ŒI D 0), both ˛ D 1 and ˛ 0 D 1, and we
again find the Michaelis–Menten equation described earlier.
Just like the Michaelis–Menten equation, this equation can be rearranged to fit
a double-reciprocal plot:

1 ˛0 Km  ˛ 1
D C 
V0 Vmax Vmax ŒS

If ˛ > 1 and ˛ 0 > 1, the inhibition is mixed. For competitive inhibition ˛ 0 D 1

and for uncompetitive inhibition ˛ D 1. Thus, three different equations are ob-
tained for the three different types of inhibition, and a Lineweaver–Burk plot of the
kinetic data can reveal the type of inhibition that the inhibitor performs (Fig. 4.9).

Competitive Inhibition

The double-reciprocal equation for competitive inhibition is as follows:

1 ˛0 Km  ˛ 1
D C 
V0 Vmax Vmax ŒS
76 4 Enzyme Kinetics

Fig. 4.10 Lineweaver–Burk plot showing competitive inhibition. Increasing the concen-
tration of the inhibitor [I] increases the steepness of the slope but does not (significantly)
change the y-intercept

Where
ŒI
˛ D1C
KI
Based on this equation, a double-reciprocal plot should give a straight line, with
the intercept 1=Vmax and slope Km ˛=Vmax . Different Lineweaver–Burk plots with
varying inhibitor concentrations should, therefore, give different slopes (because
˛ increases with the inhibitor concentration) but the same y-intersect (Fig. 4.10).
This means, that V max at different concentrations of a competitive inhibitor is un-
changed. However, the apparent K m , K m,app (Km;app D Km  ˛), differs. If double-
reciprocal plots of 1 / V 0 against 1 / [S] with varied inhibitor concentrations yield
straight lines, with different slopes but with the same y-intersect, the inhibitor is
competitive.

Calculating K I , K m and V max

If the inhibitor is competitive, only one inhibitor constant needs to be calculated.
To calculate the inhibitor constant, several assays with increasing concentrations
of substrates but different initial inhibitor concentrations must be conducted. Each
4.2 Enzyme Kinetics Theory Content 77

Fig. 4.11 Using

Lineweaver–Burk plots
to calculate Km /V max .
Plotting the slopes of the
Lineweaver–Burk plots for
each inhibitor concentration
[I] allows a linear regres-
sion which y-intersect will
provide K m / V max

of the resulting datasets should be plotted, and the slopes and y-intersects can be
determined by linear regression. From these fits, V max can be calculated as the re-
ciprocal of the y-intercept. If none of the kinetic parameters have been determined,
this linear fit does not provide enough information to determine K I and Km . To
determine these parameters, it is necessary to plot the “slopes” (Km  ˛=Vmax) from
the different assays against the inhibitor concentration [I], based on the following
equation:
Km  ˛ Km Km 1
slopecompetitive D D C   ŒI
Vmax Vmax Vmax KI
This plot should, therefore, also result in a straight line with the inter-
sect Km =Vmax and slope .Km =Vmax /  1=KI . We already know V max from
the Lineweaver–Burk plot, so with the y-intersect, we can calculate Km D
y-intersect  Vmax (Fig. 4.11).
If we measure the slope Km =Vmax  1=KI , we now know V max and K m , so we
can calculate KI D Km =.Vmax  slope/ D y-intersect=slope.

Calculating the Kinetic Parameters When Using a Competitive

Inhibitor

 Set up kinetics experiments using increasing concentrations of substrate [S] and

repeat for different initial concentrations of inhibitors [I].
 Prepare Lineweaver–Burk plots of the kinetic data and fit the data using linear
regression (one fit per inhibitor concentration).
 The y-intersects of the Lineweaver–Burk plots at different inhibitor concentra-
tions should be the same (or at least close).
78 4 Enzyme Kinetics

 Take the reciprocal to the y-intersect; this is V max .

 Plot the slopes of each of these lines as a function of the inhibitor concentration
in a new plot and fit this plot using linear regression.
 To calculate K m , multiply the y-intersect of this line with V max .
 To calculate K I , divide the y-intersect of this line with the slope.

Uncompetitive Inhibition

An uncompetitive inhibitor interacts with the enzyme-substrate complex but not

with the enzyme alone. For uncompetitive inhibition, the double-reciprocal equa-
tion is as follows:
1 ˛0 Km 1
D C 
V0 Vmax Vmax ŒS

Fig. 4.12 Lineweaver–Burk plot showing uncompetitive inhibition. Increasing the con-
centration of the inhibitor [I] does not alter the slope of the plot but does increase the y-
intercept
4.2 Enzyme Kinetics Theory Content 79

Where
ŒI
˛0 D 1 C
KI0
This equation shows that a double-reciprocal plot of enzyme kinetic data with
varying concentrations of an uncompetitive inhibitor should give a straight line
with varying y-intersects but with the same slopes (Fig. 4.12). In enzyme kinetic
assays with an uncompetitive inhibitor, the apparent K m and V max will change with
increasing inhibitor concentrations.

Calculating KI0 , K m and V max

When working with an uncompetitive inhibitor, no parameters can be calculated
from the initial Lineweaver–Burk plots. To calculate the different parameters, the
“y-intersects” of these plots must be plotted against the inhibitor concentration:

˛0 1 1 1
y-intersectcompetitive D D C   ŒI
Vmax Vmax Vmax KI

This plot should, therefore, result in a straight line with the intercept 1/max, and
slope 1=.Vmax  KI /:Vmax can be then simply calculated from this fit, by taking the
reciprocal to the y-intercept. Once we know V max , K I can therefore be calculated.
The slope is equal to 1=.Vmax  KI / so Ki D 1=.Vmax  slope/ D y-intersect=slope.
Because the initial Lineweaver–Burk plots have the same slopes as without in-
hibitors, Km =Vmax ; Km can now be calculated by multiplying these slopes with
V max obtained from the plot of y-intersects against inhibitor concentrations.

Calculating the Kinetic Parameters When Using an Uncompetitive

Inhibitor:

 Set up kinetics experiments using increasing concentrations of substrate [S] and

repeat for different initial concentrations of inhibitors [I].
 Prepare Lineweaver–Burk plots of the kinetic data and fit the data using linear
regression (1 fit per inhibitor concentration).
 Plot the y-intersects of each of these fits as a function of the inhibitor concen-
tration [I].
 To calculate V max take the reciprocal of the y-intersect of this plot.
 To calculate K I divide the y-intersect of this plot with the slope.
 To calculate K m go back to the first Lineweaver–Burk plots, take the slope from
one of these plots (the slope should be the same, or close, for all the plots), and
multiply it with V max .
80 4 Enzyme Kinetics

Mixed/Non-competitive Inhibition

A mixed inhibitor interacts with the enzyme alone and with the enzyme-substrate
complex. The double-reciprocal equation for mixed inhibition is as follows:

1 ˛0 Km  ˛ 1
D C 
V0 Vmax Vmax ŒS

where
ŒI
˛ D1C
KI
and
ŒI
˛0 D 1 C
KI0
For mixed inhibition, the Lineweaver–Burk plots show both different slopes
and different y-intersects at different inhibitor concentrations. To calculate the
parameters in this case, two new plots must be prepared. First, plot the intersects
against the inhibitor concentrations; this makes it possible to obtain KI0 and V max

Fig. 4.13 Lineweaver–Burk plot showing mixed inhibition. Increasing the concentration
of the inhibitor [I] increases the steepness of the slope and also increases the y-intercept
4.2 Enzyme Kinetics Theory Content 81

as described under uncompetitive inhibition (Fig. 4.13). Secondly, plot the slopes
against the inhibitor concentrations from this, K i can be found as explained in the
competitive inhibition. The slope of this plot is Km =Vmax, therefore, multiplying
this slope with V max already obtained gives K m .

Non-competitive Inhibition
In the special case of mixed inhibition where ˛ D ˛ 0 , i.e., KI D KI0 , the type
of inhibition is called non-competitive inhibition. In this case, the inhibitor in-
teracts favorably with the enzyme-substrate complex as it does with the enzyme
alone. When plotting kinetic data in a Lineweaver–Burk plot, a common x-inter-
sect shows that the competitor is non-competitive.
The double-reciprocal equation for non-competitive inhibition is thus as fol-
lows:
1 ˛0 Km  ˛ 1
D C 
V0 Vmax Vmax ŒS
where
ŒI
˛ D1C
KI
When plotting kinetic data using a non-competitive inhibitor, the apparent K m
remains the same as the actual K m , and it can be calculated from a Lineweaver–
Burk plot by dividing the slope with the y-intersect. To calculate V max and K I , the
y-intersects of the different lines obtained from linear regression of Lineweaver–
Burk plots at different inhibitor concentrations must be plotted against the inhibitor
concentration. When fitted using linear regression, V max and K I can be calculated
from this plot in the same manner as in the case of uncompetitive inhibition: V max
is calculated by taking the reciprocal to the y-intersect of this line, and K I is calcu-
lated by dividing the y-intersect with the slope.

Calculating the Kinetic Parameters When Using a Non-Competitive

Inhibitor:

 Set up kinetics experiments using increasing concentrations of substrate [S] and

repeat for different initial concentrations of inhibitors [I].
 Prepare Lineweaver–Burk plots of the kinetic data and fit the data using linear
regression (1 fit per inhibitor concentration).
 Calculate K m by dividing the slope of any of these lines with the corresponding
y-intersect (K m obtained should not depend on the line used).
82 4 Enzyme Kinetics

 Plot the y-intersects of each of these fits as a function of the inhibitor concen-
tration.
 To calculate V max , take the reciprocal of the y-intersect of this plot.
 To calculate K I , divide the y-intersect of this plot with the slope.

Methanol Poisoning

To give a real-world example of enzyme inhibition, we can use methanol poisoning

and its treatment. As discussed previously ADH is not completely specific for
ethanol. It also catalyzes the formation of aldehydes from other alcohols. One
of these alcohols is methanol, which is metabolized into formaldehyde and other
toxic compounds that can cause blindness or death. Methanol poisoning is quite
common and can be caused by the ingestion of homemade alcohol. Methanol and
ethanol are thus competitive substrates, and ethanol is actually used to prevent
poisoning after the ingestion of methanol, because it inhibits ADH in catalyzing
methanol, preventing the formation of harmful products and giving time for the
methanol to be excreted. In this case, ethanol acts as a competitive inhibitor.

Master Mixes

As you can see, enzymes are incredibly sensitive to work with, and measuring their
activity is a precise science. As such, we need to minimize any potential errors we
can introduce into our measurements. One such way is to use master mixes when
preparing solutions.
While the pipettes you use in the lab are incredibly accurate, they always feature
a small degree of error, which comes into play every time you pipette a liquid.
This error can be especially problematic when working with very small volumes.
For example, a 0.1 µL error when pipetting 1 mL of liquid represents 1 / 10,000th

Fig. 4.14 Theory of master mixes. By using a master mix the number of pipette move-
ments and, thus, error is reduced. In the above example, we need to mix three stock reagents
at a standard concentration with one reagent at different concentrations in each tube. a If
we pipette each reagent individually for five tubes we will have to pipette 20 times. b If
we create a master mix of the three standard reagents, the number of pipette movements is
reduced to 13. The more samples, the greater the benefits of using a master mix
I
4.2 Enzyme Kinetics Theory Content 83
84 4 Enzyme Kinetics

of the total volume and so is negligible. However, if you were pipetting 0.5 µL,
then an error of 0.1 µL (1/5th of the volume) could have profound effects on your
experiment.
Therefore, to minimize this error, we use master mixes. Rather than pipetting
multiple individual reagents one at a time, we can join similar pipette movements
together, as shown in Fig. 4.14.
The simple act of reducing the frequency pipette movements reduces the risk
of error, but, further, by working with larger volumes we also reduce the overall
impact. Also, as an added bonus, it saves your thumb muscles from too much
pipetting!

Calculating Substrate Concentrations

In the virtual experiment, you will need to calculate how much substrate to add to
a tube to obtain the desired substrate concentration. This can be done using the
following formula:
C1  V 1 D C2  V 2
where C = concentration and V = volume. Remember to use the same units on
both sides of the equals sign!
Example: We have a stock solution of substrate at 1 M and we need a final
concentration of 200 mM in a volume of 500 µL. What is the volume of stock
solution that we need to add to achieve the desired concentration?
If the stock concentration is C1, C2 will be the desired final concentration, and
V2 will be the final volume. This makes V1 the unknown element. We can now
isolate V1, insert the known values, and calculate the volume:

V 1 D .C 2  V 2/=C 1
V 1 D .0:2 M  0:0005 L/=1 M D 0:0001 L D 100 L

Spectrophotometer

A spectrophotometer is an instrument that permits the determination of the ratio

between the intensity of the light emitted from a lamp in the spectrophotometer
and the light that passes through a given solution. This ratio can be used to de-
termine the concentration of the molecules in the solution. The spectrophotometer
is set only to measure at a certain wavelength. This wavelength can be adjusted
4.3 Let’s Get Started 85

depending on the compound, so that the optimal wavelength for measuring is used.
The spectrophotometer displays the so-called absorbance (A), which is calculated
as log(I 0 / I t ), where I 0 is the intensity of the incident light, and this is the inten-
sity of the light that passes through the solution which is actually measured by the
spectrophotometer.
For kinetics assays, you want to measure the specific wavelength relative to the
product formed. To start, prepare a mix of substrate, buffer, and potential inhibitor
and then transfer to a spectrophotometer cuvette. Begin recording here, as this will
provide your baseline reading. Upon adding the enzyme to the cuvette the reaction
will start immediately, and any changes will be measured by the spectrophotome-
ter. If you miss these critical early data points, then you will not be able to measure
the initial rate V 0 , which is necessary to calculate all other kinetic parameters.

4.3 Let’s Get Started

And now your introduction to biochemistry is almost at a close. You’ve covered

how various macromolecules are formed, with a major focus on carbohydrates, and
then dived even deeper inside the molecules to look at ionic and covalent bonding
at the molecular level. In the Enzyme Kinetics simulation, you will get to see how
some of these molecules are formed in our body, and the key role enzymes play in
this.

Techniques Used in the Lab

 Spectrophotometry
 Data analysis of enzyme kinetics measurements

Learning Objectives
At the end of this simulation, you will be able to . . .

 Design enzyme kinetics experiments

 Describe the Michaelis–Menten model of enzyme kinetics
 Analyze spectrophotometer data and calculate Km and Vmax
 Visualize how enzyme kinetics can be modified by genetic mutations
 Test several inhibitors to learn about inhibition kinetics
86 4 Enzyme Kinetics

ACCESS THE VIRTUAL LAB SIMULATION HERE www.labster.com/

springer BY USING THE UNIQUE CODE AT THE END OF THE PRINTED
BOOK. IF YOU USE THE E-BOOK YOU CAN PURCHASE ACCESS TO
THE SIMULATIONS THROUGH THE SAME LINK.

Further Reading
Alberts B et al (2015) The molecular biology of the cell, 6th edn. Garland Science, Abingdon
Nelson DL, Cox MM, Lehninger AL (2013) Lehninger Principles of Biochemistry, 6th edn.
W.H. Freeman, New York
OpenStax CNX (2018) OpenStax, biology. http://cnx.org/contents/185cbf87-c72e-48f5-
[email protected]. Accessed 1 June 2018
Urey LA et al (2014) Campbell biology, 10th edn. Pearson, Boston
 springer-spektrum.de

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