Chapter 1

Cell structure and function dec 16

1.1 Lesson 3.1: Introduction to Cells

Lesson Objectives
• Identify the scientists that first observed cells.
• Outline the importance of microscopes in the discovery of cells.
• Summarize what the cell theory proposes.
• Identify the limitations on cell size.
• Identify the four parts common to all cells.
• Compare prokaryotic and eukaryotic cells.

Introduction

Knowing the make up of cells and how cells work is necessary to all of the biological sciences. Learning
about the similarities and differences between cell types is particularly important to the fields of cell biology
and molecular biology. The importance of the similarities and differences between cell types is a unifying
theme in biology. They allow the principles learned from studying one cell type to be applied when learning
about other cell types. For example, learning about how single-celled animals or bacteria work can help
us understand more about how human cells work. Research in cell biology is closely linked to genetics,
biochemistry, molecular biology, and developmental biology.

Discovery of Cells

A cell is the smallest unit that can carry out the processes of life. It is the basic unit of all living things, and
all organisms are made up of one or more cells. In addition to having the same basic structure, all cells carry
out similar life processes. These include transport of materials, obtaining and using energy, waste disposal,
replication, and responding to their environment.
If you look at living organisms under a microscope you will see they are made up of cells. The word cell
was first used by Robert Hooke, a British biologist and early microscopist. Hooke looked at thin slices of

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cork under a microscope. The structure he saw looked like a honeycomb as it was made up of many tiny
units. Hooke’s drawing is shown in Figure 1.1. In 1665 Hooke published his book Micrographia, in which
he wrote:

... I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but
that the pores of it were not regular.... these pores, or cells, ... were indeed the first microscopical
pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that
had made any mention of them before this...

Figure 1.1: Drawing of the structure of cork from as it appeared under the microscope to Robert Hooke.
The first scientific use of the word appears in this book.

During the 1670s, the Dutch tradesman Antony van Leeuwenhoek, shown in Figure 1.2, used microscopes
to observe many microbes and body cells. Leeuwenhoek developed an interest in microscopy and ground
his own lenses to make simple microscopes. Compound microscopes, which are microscopes that use more
than one lens, had been invented around 1595. Several people, including Robert Hooke, had built compound
microscopes and were making important discoveries with them during Leeuwenhoek’s time. These compound
microscopes were very similar to the microscopes in use today. However, Leeuwenhoek was so good at making
lenses that his simple microscopes were able to magnify much more clearly than the compound microscopes
of his day. His microscope’s increased ability to magnify over 200 times is comparable to a modern compound
light microscope.
Leeuwenhoek was also very curious, and he took great care in writing detailed reports of what he saw under
his microscope. He was the first person to report observations of many microscopic organisms. Some of his
discoveries included tiny animals such as ciliates, foraminifera, roundworms, and rotifers, shown in Figure
1.3. He discovered blood cells and was the first person to see living sperm cells. In 1683, Leeuwenhoek wrote
to the Royal Society of London about his observations on the plaque between his own teeth, ”a little white
matter, which is as thick as if ’twere batter.” He called the creatures he saw in the plaque animacules, or
tiny animals. This report was among the first observations on living bacteria ever recorded.

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Figure 1.2: Antony van Leeuwenhoek (1632-1723). His carefully crafted microscopes and insightful observa-
tions of microbes led to the title the "Father of Microscopy."

Figure 1.3: Rotifers, similar to the type that Leeuwenhoek saw under his microscope.

Microscopes

Hooke’s and Leeuwenhoek’s studies and observations filled people with wonder because their studies were
of life forms that were everywhere, but too small to see with the naked eye. Just think how amazed you
would be if you were to read about the first accounts of a newly discovered microorganism from the moon
or Mars. Your first thought might be ”Things can live there?!” which was probably the first thought of the
people who read Hooke’s and Leeuwenhoek’s accounts. The microscope literally opened up an amazing new
dimension in the natural sciences, and became a critical tool in the progress of biology.
Magnifying glasses had been in use since the 1300s, but the use of lenses to see very tiny objects was a
slowly-developing technology. The magnification power of early microscopes was very limited by the glass
quality used in the lenses and the amount of light reflected off the object. These early light microscopes
had poor resolution and a magnification power of about 10 times. Compare this to the over 200 times

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magnification that Leeuwenhoek was able to achieve by carefully grinding his own lenses. However, in time
the quality of microscopes was much improved with better lighting and resolution. It was through the use
of light microscopes that the first discoveries about the cell and the cell theory (1839) were developed.
However, by the end of the 19th century, light microscopes had begun to hit resolution limits. Resolution
is a measure of the clarity of an image; it is the minimum distance that two points can be separated by and
still be distinguished as two separate points. Because light beams have a physical size, it is difficult to see
an object that is about the same size as the wavelength of light. Objects smaller than about 0.2 micrometers
appear fuzzy, and objects below that size just cannot be seen. Light microscopes were still useful, but most
of the organelles and tiny cell structures discussed in later lessons were invisible to the light microscope.

Figure 1.4: Left to right: (a) Hookes light microscope (b) Modern electron microscope.

In the 1950s, a new system was developed that could use a beam of electrons to resolve very tiny dimensions
at the molecular level. Electron microscopes, one of which is shown in Figure 1.4, have been used to produce
images of molecules and atoms. They have been used to visualize the tiny sub-cellular structures that were
invisible to light microscopes. Many of the discoveries made about the cell since the 1950s have been made
with electron microscopes.

The Cell Theory

Later, biologists found cells everywhere. Biologists in the early part of the 19th century suggested that all
living things were made of cells, but the role of cells as the primary building block of life was not discovered
until 1839 when two German scientists, Theodor Schwann, a zoologist, and Matthias Jakob Schleiden, a
botanist, suggested that cells were the basic unit of all living things. Later, in 1858, the German doctor
Rudolf Virchow observed that cells divide to produce more cells. He proposed that all cells arise only from

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other cells. The collective observations of all three scientists form the cell theory. The modern cell theory
states that:

• All organisms are made up of one or more cells.

• All the life functions of an organism occur within cells.
• All cells come from preexisting cells.

As with any theory, the cell theory is based on observations that over many years upheld the basic conclusions
of Schwann’s paper written in 1839. However, one of Schwann’s original conclusions stated that cells formed
in a similar way to crystals. This observation, which refers to spontaneous generation of life, was discounted
when Virchow proposed that all cells arise only from other cells. The cell theory has withstood intense
examination of cells by modern powerful microscopes and other instruments. Scientists use new techniques
and equipment to look into cells to discover additional explanations for how they work.

Diversity of Cells

Different cells within a single organism can come in a variety of sizes and shapes. They may not be very big,
but their shapes can be very different from each other. However, these cells all have common abilities, such
as getting and using food energy, responding to the external environment, and reproducing. A cell’s shape
determines its function.

Cell Size

If cells have such an important job, why are they so small? And why are there no organisms with huge
cells? The answers to these questions lie in a cell’s need for fast, easy food. The need to be able to pass
nutrients and gases into and out of the cell sets a limit on how big cells can be. The larger a cell gets, the
more difficult it is for nutrients and gases to move in and out of the cell.
As a cell grows, its volume increases more quickly than its surface area. If a cell was to get very large, the
small surface area would not allow enough nutrients to enter the cell quickly enough for the cell’s needs.
This idea is explained in Figure 1.5. However, large cells have a way of dealing with some size challenges.
Big cells, such as some white blood cells, often grow more nuclei so that they can supply enough proteins
and RNA for the cell’s needs. Large, metabolically active cells often have lots of folds in their cell surface
membrane. These folds increase the surface area available for transport into or out of the cell. Such cell
types are found lining your small intestine, where they absorb nutrients from your food through little folds
called microvilli.
Scale of Measurements
1 centimeter (cm) = 10 millimeters (mm) = 10-2 meters (m)
1 mm = 1000 micrometers (µm) = 10-3 m
1 µm = 1000 nanometers (nm) = 10-6 m
1 nm = 10-3 µm

Imagine cells as little cube blocks. A small cube cell is one unit in length.

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Figure 1.5: A small cell (left), has a larger surface-area to volume ratio than a bigger cell (center). The
greater the surface-area to volume ratio of a cell, the easier it is for the cell to get rid of wastes and take in
essential materials such as oxygen and nutrients.

The total surface area of this cell is calculated by the equation:

height × width × number of sides × number of boxes
1×1×6×1=6
The volume of the cell is calculated:
height x width x length x number of boxes
1×1×1×1=1
The surface-area to volume ratio is:
area ÷ volume
6 ÷ 1=6
A larger cell that is 3 units in length would have a total surface area of
3 × 3 × 6 × 1 = 54
and a volume of:
3 × 3 × 3 × 1 = 27
The surface-area to volume ratio of the large cell is:
54÷ 27=2
Now, replace the three unit cell with enough one unit cells to equal the volume of the single three unit cell.
This can be done with 27 one unit cells. Find the total surface area of the 27 cells:
1 × 1 × 6 × 27 = 162 units
The total volume of the block of 27 cells is:
1 × 1 × 1 × 27 = 27
The surface-area to volume ratio of the 27 cells is:
162 ÷ 27=6
An increased surface area to volume ratio means increased exposure to the environment. This means that

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nutrients and gases can move in and out of a small cell more easily than in and out of a larger cell.
The smallest prokaryotic cell currently known has a diameter of only 400 nm. Eukaryotic cells normally
range between 1– 100 µm in diameter.

Figure 1.6: Ostrich eggs (a) can weigh as much as 1.5 kg, and be 13 cm in diameter, whereas each of the
mouse cells (b) shown at right are each about 10 m in diameter, much smaller than the period at the end of
this sentence.

The cells you have learned about so far are tinier than the period at the end of this sentence, so they are
normally measured on a very tiny scale. Most cells are between 1 and 100 µm in diameter. The mouse cells
in Figure 1.6 are about 10 µm in diameter. One exception however, is eggs. Eggs contain the largest known
single cell, and the ostrich egg is the largest of them all. The ostrich egg in Figure 1.6 is over 10,000 times
larger than the mouse cell.

Cell Shape

The variety of cell shapes seen in prokaryotes and eukaryotes reflects the functions that each cell has. Each
cell type has evolved a shape that best helps it survive and do its job. For example, the nerve cell in Figure
1.7 has long, thin extensions that reach out to other nerve cells. The extensions help the nerve cell pass
chemical and electrical messages quickly through the body. The spikes on the pollen grain help it stick to a
pollinating insect or animal so that it can be transferred to and pollinate another flower. The long whip-like
flagella (tails) of the algae Chlamydomonas help it swim in water.

Parts of a Cell

There are many different types of cells, but all cells have a few things in common. These are:

• a cell or plasma membrane

• cytoplasm

• ribosomes for protein synthesis

• DNA (genetic information)

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Figure 1.7: Cells come in very different shapes. Left to right, top row: Long, thin nerve cells; biconcave red
blood cells; curved-rod shaped bacteria. Left to right, bottom row: oval, flagellated algae and round, spiky
pollen grains are just a sample of the many shapes.

The cell membrane is the physical boundary between the inside of the cell (intracellular) and its outside
environment (extracellular). It acts almost like the ”skin” of the cell. Cytoplasm is the general term for all of
the material inside the cell. Cytoplasm is made up of cytosol, a watery fluid that contains dissolved particles
and organelles. Organelles are structures that carry out specific functions inside the cell. Ribosomes are
the organelles on which proteins are made. Ribosomes are found throughout the cytosol of the cell. All cells
also have DNA. DNA contains the genetic information needed for building structures such as proteins and
RNA molecules in the cell.

Two Types of Cells

There are two cell types: prokaryotes and eukaryotes. Prokaryotic cells are usually single-celled and smaller
than eukaryotic cells. Eukaryotic cells are usually found in multicellular organisms, but there are some
single-celled eukaryotes.

Prokaryotic Cells

The bacterium in Figure 1.8 is a prokaryote. Prokaryotes are organisms that do not have a cell nucleus
nor any organelles that are surrounded by a membrane. Some cell biologists consider the term ”organelle” to
describe membrane-bound structures only, whereas other cell biologists define organelles as discrete structures
that have a specialized function. Prokaryotes have ribosomes, which are not surrounded by a membrane but
do have a specialized function, and could therefore be considered organelles. Most of the metabolic functions
carried out by a prokaryote take place in the plasma membrane.
Most prokaryotes are unicellular and have a cell wall that adds structural support and acts as a barrier

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Figure 1.8: Diagram of a typical prokaryotic cell. Among other things, prokaryotic cells have a plasma
membrane, cytoplasm, ribosomes, and DNA. Prokaryotes do not have membrane-bound organelles or a cell
nucleus.

against outside forces. Some prokaryotes have an extra layer outside their cell wall called a capsule, which
helps them stick to surfaces or to each other. Prokaryotic DNA usually forms a circular molecule and is found
in the cell’s cytoplasm along with ribosomes. Prokaryotic cells are very small; most are between 1–10 µm in
diameter. They are found living in almost every environment on Earth. Biologists believe that prokaryotes
were the first type of cells on Earth and that they are the most common organisms on Earth today.

Eukaryotic Cells

A eukaryote is an organism whose cells are organized into complex structures by internal membranes and
a cytoskeleton, as shown in Figure 1.12. The most characteristic membrane-bound structure of eukaryotes
is the nucleus. This feature gives them their name, which comes from Greek and means ”true nucleus.” The
nucleus is the membrane-enclosed organelle that contains DNA. Eukaryotic DNA is organized in one or
more linear molecules, called chromosomes. Some eukaryotes are single-celled, but many are multicellular.
In addition to having a plasma membrane, cytoplasm, a nucleus and ribosomes, eukaryotic cells also contain
membrane-bound organelles. Each organelle in a eukaryote has a distinct function. Because of their com-
plex level of organization, eukaryotic cells can carry out many more functions than prokaryotic cells. The
main differences between prokaryotic and eukaryotic cells are shown in Figure 1.11 and listed in Table 1.
Eukaryotic cells may or may not have a cell wall. Plant cells generally have cell walls, while animal cells do
not.
Eukaryotic cells are about 10 times the size of a typical prokaryote; they range between 10 and 100 µm in
diameter while prokaryotes range between 1 and 10 µm in diameter, as shown in Figure 1.10. Scientists
believe that eukaryotes developed about 1.6 – 2.1 billion years ago. The earliest fossils of multicellular
organisms that have been found are 1.2 billion years old.

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Figure 1.9: A eukaryotic cell, represented here by a model animal cell is much more complex than a prokary-
otic cell. Eukaryotic cells contain many organelles that do specific jobs. No single eukaryotic cell has all the
organelles shown here, and this model shows all eukaryotic organelles.

Figure 1.10: The relative scale of prokaryotic and eukaryotic cells. See how eukaryotic cells are generally 10
to 100 times larger than prokaryotic cells.

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Figure 1.11: The main differences between prokaryotic and eukaryotic cells. Eukaryotic cells have membrane
bound organelles while prokaryotic cells do not.

Table 1.1: Structural Differences Between Prokaryotic Cells and Eukaryotic Cells

Presence of Prokaryote Eukaryote

yes yes
Plasma membrane
Genetic material (DNA) yes yes
Cytoplasm yes yes
Ribosomes yes yes
Nucleus no yes
Nucleolus no yes
Mitochondria no yes
Other membrane-bound or- no yes
ganelles
Cell wall yes some (not around animal cells)
Capsule yes no
Average diameter 0.4 to 10 µm 1 to 100 µm

Lesson Summary
• Robert Hooke first saw and named cells. Antony van Leeuwenhoek was the first person to see living
cells.
• Before the development of microscopes, the existence of cellular life was unknown. The development of
light microscopes and later electron microscopes helped scientists learn more about the cell. Most of the
discoveries about cell structure since the 1950s have been made due to the use of electron microscopes.
• The cell theory states that all living things are made of one or more cells, that cells are the basic unit
of life, and that cells come only from other cells.
• Cell size is limited by a cell’s surface area to volume ratio. A cell’s shape is determined by its function.
• Parts common to all cells are the plasma membrane, the cytoplasm, ribosomes, and genetic material.
• Prokaryotic cells lack a nucleus and other membrane-bound organelles.

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Review Questions
1. Describe the contributions of Hooke and Leeuwenhoek to cell biology.

2. What enabled Leeuwenhoek to observe things that nobody else had seen before?

3. What three things does the cell theory propose?

4. A cell has a volume of 64 units, and total surface area of 96 units. What is the cell’s surface area to
volume ratio (surface area ÷ volume)?

5. What is the relationship between cell shape and function?

6. What are the three basic parts of a cell?

7. Compare prokaryotic and eukaryotic cells. Identify two differences between prokaryotic and eukaryotic
cells.

8. Is the cell in this image prokaryotic or eukaryotic? Explain your answer.

Figure 1.12

Further Reading / Supplemental Links

• Human Anatomy © 2003 Martini, Timmons, Tallitsch. Published by Prentice Hall, Inc.

• http://www.ucmp.berkeley.edu/history/hooke.htmlhttp://www.ucmp.berkeley.edu/history/hooke.html

• http://www.ucmp.berkeley.edu/history/leeuwenhoek.htmlhttp://www.ucmp.berkeley.edu/history/leeuwenhoek.html

• http://fig.cox.miami.edu/˜cmallery/150/unity/cell.text.htmhttp://fig.cox.miami.edu/˜cmallery/150/unity/cell.text.htm

• http://en.wikibooks.org/wiki/Cell_Biology/Historyhttp://en.wikibooks.org/wiki/Cell_Biology/History

• http://en.wikibooks.org/wiki/General_Biology/Cellshttp://en.wikibooks.org/wiki/General_Biology/Cells

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 • http://www.ucmp.berkeley.edu/history/hooke.htmlhttp://www.ucmp.berkeley.edu/history/hooke.html

• http://www.brianjford.com/wav-mict.htmhttp://www.brianjford.com/wav-mict.htm

• http://fig.cox.miami.edu/˜cmallery/150/unity/cell.text.htmhttp://fig.cox.miami.edu/˜cmallery/150/unity/cell.text.htm

• http://www.cellsalive.com/toc.htmhttp://www.cellsalive.com/toc.htm

• http://publications.nigms.nih.gov/insidethecell/index.htmlhttp://publications.nigms.nih.gov/insidethecell/index.html

• http://cellimages.ascb.org/cdm4/browse.php?CISOROOT=/p4041coll11http://cellimages.ascb.org/cdm4/browse.php?

• http://en.wikipedia.orghttp://en.wikipedia.org

• http://www.flashcardmachine.com/

Vocabulary
cell The smallest unit that can carry out the processes of life; the basic unit of all living things.

cell membrane The physical boundary between the inside of the cell (intracellular) and its outside envi-
ronment (extracellular).

cytoplasm The general term for all of the material inside the cell, between the cell membrane and the
nucleus.

cytosol A watery fluid that contains dissolved particles and organelles; makes up cytoplasm.

DNA Deoxyribonucleic acid, the genetic material; contains the genetic information needed for building
structures such as proteins.

eukaryote An organism whose cells are organized into complex structures by internal membranes and a
cytoskeleton.

eukaryotic cells Typical of multi-celled organisms; have membrane bound organelles; usually larger than
prokaryotic cells.

nucleus The membrane bound organelle that contains DNA; found in eukaryotic cells.

organelle Structure that carries out specific functions inside the cell.

prokaryotic cells Typical of simple, single-celled organisms, such as bacteria; lack a nucleus and other
membrane bound organelles.

resolution A measure of the clarity of an image; the minimum distance that two points can be separated
by and still be distinguished as two separate points.

ribosomes The organelles on which proteins are made (synthesized).

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Points to Consider

Next we focus on cell structures and their roles.

• What do you think is the most important structure in a cell? Why?

• Using each of the functions of the four common cell parts, state why each one is necessary for the
survival of the cell.

Lesson 3.2: Cell Structures

Lesson Objectives
• Outline the structure of the plasma membrane.

• Distinguish cytoplasm from cytosol.

• Name three types of protein fibers that make up the cytoskeleton.

• Compare and and contrast plant and animal cells

• List three major organelles found only in eukaryotic cells and identify their roles.

• Given medical conditions, determine what cell’s structure and function is affected and why.

• Be able to relate all organelles involved in the production of proteins

Introduction

The invention of the microscope opened up a previously unknown world. Before the invention of the mi-
croscope, very little was known about what made up living things and non-living things, or where living
things came from. During Hooke’s and Leeuwenhoek’s time, spontaneous generation — the belief that living
organisms grow directly from decaying organic substances — was the accepted explanation for the appear-
ance of small organisms. For example, people accepted that mice spontaneously appeared in stored grain,
and maggots formed in meat with no apparent external influence. Once cells were discovered, the search for
answers to such questions as ”what are cells made of?” and ”what do they do?” became the focus of study.

Cell Function

Cells share the same needs: the need to get energy from their environment, the need to respond to their
environment, and the need to reproduce. Cells must also be able to separate their relatively stable interior
from the ever-changing external environment. They do this by coordinating many processes that are carried
out in different parts of the cell. Structures that are common to many different cells indicate the common
history shared by cell-based life. Examples of these common structures include the components of both the
cell (or plasma) membrane and the cytoskeleton, and other structures shown in Figure 1.13.

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Figure 1.13: The structure and contents of a typical animal cell. Every animal cell has a cell membrane,
cytoplasm, and a nucleus, but not all cells have every structure shown here. For example, some cells such
as red blood cells do not have any mitochondria, yet others such as muscle cells may have thousands of
mitochondria.

Plasma Membrane

The plasma membrane (also called the cell membrane) has many functions. For example, it separates the
internal environment of the cell from the outside environment. It allows only certain molecules into and
out of the cell. The ability to allow only certain molecules in or out of the cell is referred to as selective
permeability or semipermeability. The plasma membrane also acts as the attachment point for both the
intracellular cytoskeleton and, if present, the cell wall.
The plasma membrane is a lipid bilayer that is common to all living cells. A lipid bilayer is a double
layer of closely-packed lipid molecules. The membranes of cell organelles are also lipid bilayers. The plasma
membrane contains many different biological molecules, mostly lipids and proteins. These lipids and proteins
are involved in many cellular processes.

Phospholipids

The main type of lipid found in the plasma membrane is phospholipid. A phospholipid is made up of a polar,
phosphorus-containing head, and two long fatty acid, non-polar ”tails.” That is, the head of the molecule
is hydrophilic (water-loving), and the tail is hydrophobic (water-fearing). Cytosol and extracellular fluid
are made up of mostly water. In this watery environment, the water loving heads point out towards the
water, and the water fearing tails point inwards, and push the water out. The resulting double layer is
called a phospholipid bilayer. A phospholipid bilayer is made up of two layers of phospholipids, in which
hydrophobic fatty acids are in the middle of the plasma membrane, and the hydrophilic heads are on the
outside. An example of a simple phospholipid bilayer is illustrated in Figure 1.14.

Plasma membranes of eukaryotes contain many proteins, as well as other lipids called sterols. The proteins
have various functions, such as channels that allow certain molecules into the cell and receptors that bind
to signal molecules. In Figure 1.14, the smaller (green) molecules shown between the phospholipids are
cholesterol molecules. Cholesterol helps keep the plasma membrane firm and stable over a wide range of

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Figure 1.14: The hydrophobic fatty acids point towards the middle of the plasma membrane (pink), and
the hydrophilic heads (blue) point outwards. The membrane is stabilized by cholesterol molecules (green).
This self-organization of phospholipids results in a selectively permeable membrane which allows only certain
molecules in or out of the cell.

temperatures. At least ten different types of lipids are commonly found in plasma membranes. Each type
of cell or organelle will have a different percentage of each lipid, protein and carbohydrate.

Membrane Proteins

Plasma membranes also contain certain types of proteins. A membrane protein is a protein molecule that
is attached to, or associated with the membrane of a cell or an organelle. Membrane proteins can be put
into two groups based on how the protein is associated with the membrane.
Integral membrane proteins are permanently embedded within the plasma membrane. They have a range
of important functions. Such functions include channeling or transporting molecules across the membrane.
Other integral proteins act as cell receptors. Integral membrane proteins can be classified according to their
relationship with the bilayer:

• Transmembrane proteins span the entire plasma membrane. Transmembrane proteins are found in all
types of biological membranes.
• Integral monotopic proteins are permanently attached to the membrane from only one side.

Some integral membrane proteins are responsible for cell adhesion (sticking of a cell to another cell or
surface). On the outside of cell membranes and attached to some of the proteins are carbohydrate chains
that act as labels that identify the cell type. Shown in Figure 1.15 are two different types of membrane
proteins and associated molecules.
Peripheral membrane proteins are proteins that are only temporarily associated with the membrane.
They can be easily removed, which allows them to be involved in cell signaling. Peripheral proteins can
also be attached to integral membrane proteins, or they can stick into a small portion of the lipid bilayer
by themselves. Peripheral membrane proteins are often associated with ion channels and transmembrane
receptors. Most peripheral membrane proteins are hydrophilic.

Fluid Mosaic Model

In 1972 S.J. Singer and G.L. Nicolson proposed the now widely accepted Fluid Mosaic Model of the structure
of cell membranes. The model proposes that integral membrane proteins are embedded in the phospholipid

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Figure 1.15: Some of the membrane proteins make up a major transport system that moves molecules and
ions through the polar phospholipid bilayer.

bilayer, as seen in Figure 1.15. Some of these proteins extend all the way through the bilayer, and some
only partially across it. These membrane proteins act as transport proteins and receptors proteins.
Their model also proposed that the membrane behaves like a fluid, rather than a solid. The proteins and
lipids of the membrane move around the membrane, much like buoys in water. Such movement causes a
constant change in the ”mosaic pattern” of the plasma membrane.

Cytoplasm

The gel-like material within the cell that holds the organelles is called cytoplasm. The cytoplasm plays an
important role in a cell, serving as a ”jelly” in which organelles are suspended and held together by a fatty
membrane. The cytosol, which is the watery substance that does not contain organelles, is made up of 80%
to 90% water.
The cytosol plays a mechanical role by exerting pressure against the cell’s plasma membrane which helps
keep the shape of the cell. Cytosol also acts as the site of biochemical reactions such as anaerobic glycolysis
and protein synthesis. In prokaryotes all chemical reactions take place in the cytosol.

Cytoskeleton

The cytoskeleton is a cellular ”scaffolding” or ”skeleton” that crisscrosses the cytoplasm. All eukaryotic
cells have a cytoskeleton, and recent research has shown that prokaryotic cells also have a cytoskeleton. The
eukaryotic cytoskeleton is made up of a network of long, thin protein fibers and has many functions. It
helps to maintain cell shape. It holds organelles in place, and for some cells, it enables cell movement. The
cytoskeleton also plays important roles in both the intracellular movement of substances and in cell division.
Certain proteins act like a path that vesicles and organelles move along within the cell. The threadlike
proteins that make up the cytoskeleton continually rebuild to adapt to the cell’s constantly changing needs.
Three main kinds of cytoskeleton fibers are microtubules, intermediate filaments, and microfilaments.

• Microtubules, shown in Figure 1.16 (a), are hollow cylinders and are the thickest of the cytoskeleton
structures. They are most commonly made of filaments which are polymers of alpha and beta tubulin,
and radiate outwards from an area near the nucleus called the centrosome. Tubulin is a protein that is
composed of hollow cylinders which are made of two protein chains that are twisted around each other.
Microtubules help keep cell shape. They hold organelles in place and allow them to move around the

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 cell, and they form the mitotic spindle during cell division. Microtubules also make up parts of cilia
and flagella, the organelles that help a cell to move.
• Microfilaments, shown in Figure 1.16 (b), are made of two thin actin chains that are twisted
around one another. Microfilaments are mostly concentrated just beneath the cell membrane where
they support the cell and help keep the cell’s shape. Microfilaments form cytoplasmatic extentions
such as pseudopodia and microvilli which allows certain cells to move. The actin of the microfilaments
interacts with the protein myosin to cause contraction in muscle cells. Microfilaments are found in
almost every cell, and are numerous in muscle cells and in cells that move by changing shape such as
phagocytes (white blood cells that search the body for bacteria and other invaders).

• Intermediate filament, shown in Figure 1.16 (c), make-up differs from one cell type to another.
Intermediate filaments organize the inside structure of the cell by holding organelles and providing
strength. They are also structural components of the nuclear envelope. Intermediate filaments made
of the protein keratin are found in skin, hair, and nails cells.

Figure 1.16: The eukaryotic cytoskeleton. Microfilaments are shown in red, microtubules in green, and the
nuclei are in blue. By linking regions of the cell together, the cytoskeleton helps support the shape of the cell.
Microscopy of keratin filaments (intermediate filaments) inside cells. Microtubules in a methanol-fixated cell,
visualized with anti-beta-tubuline antibodies.

Table 1.2: Molecular structure of microtubules.Keratin intermediate filaments in skin cells (stained red).Actin
cytoskeleton of mouse embryo cells.Cytoskeleton Structure

Microtubules Intermediate Filaments Microfilaments

about 25 nm 8 to 11 nm around 7 nm
Fiber Diameter
Protein Composition tubulin, with two sub- One of different types of actin
units, alpha and beta proteins such as lamin,
tubulin vimentin, and keratin
Shape hollow cylinders made protein fiber coils two actin chains twisted
of two protein chains twisted into each other around one another
twisted around each
other

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 Table 1.2: (continued)

Microtubules Intermediate Filaments Microfilaments

Main Functions organelle and vesicle organize cell shape; po- keep cellular shape;
movement; form mitotic sitions organelles in cy- allows movement of
spindles during cell re- toplasm structural sup- certain cells by forming
production; cell motility port of the nuclear enve- cytoplasmatic exten-
(in cilia and flagella) lope and sarcomeres; in- sions or contraction of
volved in cell-to-cell and actin fibers; involved
cell-to-matrix junctions in some cell-to-cell or
cell-to-matrix junctions
Image

External Structures

Flagella (flagellum, singular) are long, thin structures that stick out from the cell membrane. Both eu-
karyotic and prokaryotic cells can have flagella. Flagella help single-celled organisms move or swim towards
food. The flagella of eukaryotic cells are normally used for movement too, such as in the movement of sperm
cells. The flagella of either group are very different from each other. Prokaryotic flagella, shown in Figure
1.17, are spiral-shaped and stiff. They spin around in a fixed base much like a screw does, which moves the
cell in a tumbling fashion. Eukaryotic flagella are made of microtubules and bend and flex like a whip.
Cilia (cilium, singular) are made up of extensions of the cell membrane that contain microtubules. Although
both are used for movement, cilia are much shorter than flagella. Cilia cover the surface of some single-celled
organisms, such as paramecium. Their cilia beat together to move the little animals through the water. In

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Figure 1.17: Bacterial flagella spin about in place, which causes the bacterial cell to "tumble."

multicellular animals, including humans, cilia are usually found in large numbers on a single surface of cells.
Multicellular animals’ cilia usually move materials inside the body. For example, the mucociliary escalator
of the respiratory system is made up of mucus-secreting cells that line the trachea and bronchi. Ciliated
cells, shown in Figure 1.18, move mucus away from the lungs. Spores, bacteria, and debris are caught in
the mucus which is moved to the esophagus by the ciliated cells, where it is swallowed.

Figure 1.18: Left: Scanning electron micrograph (SEM), of the cilia sticking up from human lung cells. Right:
Electron micrograph of cross-section of two cilia (not human), showing the positions of the microtubules
inside. Note how there are nine groups of two microtubules (called dimers) in each cilium. Each dimer is
made up of an alpha and a beta tubulin protein that are connected together.

The Nucleus and Other Organelles

The nucleus is a membrane-enclosed organelle found in most eukaryotic cells. The nucleus is the largest
organelle in the cell and contains most of the cell’s genetic information (mitochondria also contain DNA,
called mitochondrial DNA, but it makes up just a small percentage of the cell’s overall DNA content). The

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genetic information, which contains the information for the structure and function of the organism, is found
encoded in DNA in the form of genes. A gene is a short segment of DNA that contains information to
encode an RNA molecule or a protein strand. DNA in the nucleus is organized in long linear strands that
are attached to different proteins. These proteins help the DNA to coil up for better storage in the nucleus.
Think how a string gets tightly coiled up if you twist one end while holding the other end. These long
strands of coiled-up DNA and proteins are called chromosomes. Each chromosome contains many genes.
The function of the nucleus is to maintain the integrity of these genes and to control the activities of the
cell by regulating gene expression. Gene expression is the process by which the information in a gene is
”decoded” by various cell molecules to produce a functional gene product, such as a protein molecule or an
RNA molecule.
The degree of DNA coiling determines whether the chromosome strands are short and thick or long and
thin. Between cell divisions, the DNA in chromosomes is more loosely coiled and forms long thin strands
called chromatin. Before the cell divides, the chromatin coil up more tightly and form chromosomes. Only
chromosomes stain clearly enough to be seen under a microscope. The word chromosome comes from the
Greek word chroma, (color) and soma, (body) due to its ability to be stained strongly by dyes.

Nuclear Envelope

The nuclear envelope is a double membrane of the nucleus that encloses the genetic material. It separates
the contents of the nucleus from the cytoplasm. The nuclear envelope is made of two lipid bilayers, an
inner membrane and an outer membrane. The outer membrane is continuous with the rough endoplasmic
reticulum. Many tiny holes called nuclear pores are found in the nuclear envelope. These nuclear pores help
to regulate the exchange of materials (such as RNA and proteins) between the nucleus and the cytoplasm.

Nucleolus

The nucleus of many cells also contains an organelle called a nucleolus, shown in Figure 1.19. The nucleolus
is mainly involved in the assembly of ribosomes. Ribosomes are organelles made of protein and ribosomal
RNA (rRNA), and they build cellular proteins in the cytoplasm. The function of the rRNA is to provide a
way of decoding the genetic messages within another type of RNA called mRNA, into amino acids. After
being made in the nucleolus, ribosomes are exported to the cytoplasm where they direct protein synthesis.

Centrioles

Centrioles are rod-like structures made of short microtubules. Nine groups of three microtubules make up
each centriole. Two perpendicularly placed centrioles make up the centrosome. Centrioles are very important
in cellular division, where they arrange the mitotic spindles that pull the chromosome apart during mitosis.

Mitochondria

A mitochondrion (mitochondria, plural), is a membrane-enclosed organelle that is found in most eukaryotic

cells. Mitochondria are called the ”power plants” of the cell because they use energy from organic compounds
to make ATP. ATP is the cell’s energy source that is used for such things such as movement and cell division.
Some ATP is made in the cytosol of the cell, but most of it is made inside mitochondria. The number of

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Figure 1.19: The eukaryotic cell nucleus. Visible in this diagram are the ribosome-studded double membranes
of the nuclear envelope, the DNA (as chromatin), and the nucleolus. Within the cell nucleus is a viscous
liquid called nucleoplasm, similar to the cytoplasm found outside the nucleus. The chromatin (which is
normally invisible), is visible in this figure only to show that it is spread out throughout the nucleus.

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mitochondria in a cell depends on the cell’s energy needs. For example, active human muscle cells may have
thousands of mitochondria, while less active red blood cells do not have any.

Figure 1.20: Electron micrograph of a single mitochondrion within which you can see many cristae. Mito-
chondria range from 1 to 10 m in size. This model of a mitochondrian shows the organized arrangement of
the inner and outer membranes, the protein matrix, and the folded inner mitochondrial membranes.

As Figure 1.20 (a) and (b) shows, a mitochondrion has two phospholipids membranes. The smooth outer
membrane separates the mitochondrion from the cytosol. The inner membrane has many folds, called cristae.
The fluid-filled inside of the mitochondrian, called matrix, is where most of the cell’s ATP is made.
Although most of a cell’s DNA is contained in the cell nucleus, mitochondria have their own DNA. Mi-
tochandria are able to reproduce asexually and scientists think that they are descended from prokaryotes.
According to the endosymbiotic theory, mitochondria were once free-living prokaryotes that infected ancient
eukaryotic cells. The invading prokaryotes were protected inside the eukaryotic host cell, and in turn the
prokaryote supplied extra ATP to its host.

Endoplasmic Reticulum

The endoplasmic reticulum (ER) (plural, reticuli) is a network of phospholipid membranes that form
hollow tubes, flattened sheets, and round sacs. These flattened, hollow folds and sacs are called cisternae.
The ER has two major functions:

• Transport: Molecules, such as proteins, can move from place to place inside the ER, much like on an
intracellular highway.

• Synthesis: Ribosomes that are attached to ER, similar to unattached ribosomes, make proteins.
Lipids are also produced in the ER.

There are two types of endoplasmic reticulum, rough endoplasmic reticulum (RER) and smooth endoplasmic
reticulum (SER).

• Rough endoplasmic reticulum is studded with ribosomes which gives it a ”rough” appearance.
These ribosomes make proteins that are then transported from the ER in small sacs called transport

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 vesicles. The transport vesicles pinch off the ends of the ER. The rough endoplasmic reticulum works
with the Golgi apparatus to move new proteins to their proper destinations in the cell. The membrane
of the RER is continuous with the outer layer of the nuclear envelope.

• Smooth endoplasmic reticulum does not have any ribosomes attached to it, and so it has a smooth
appearance. SER has many different functions some of which are: lipid synthesis, calcium ion storage,
and drug detoxification. Smooth endoplasmic reticulum is found in both animal and plant cells and it
serves different functions in each. The SER is made up of tubules and vesicles that branch out to form
a network. In some cells there are dilated areas like the sacs of RER. Smooth endoplasmic reticulum
and RER form an interconnected network.

Figure 1.21: Image of nucleus, endoplasmic reticulum and Golgi apparatus, and how they work together.
The process of secretion from endoplasmic reticuli (orange) to Golgi apparatus (pink) is shown.

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Ribosomes

Ribosomes are small organelles and are the site of protein synthesis (or assembly). They are made of
ribosomal protein and ribosomal RNA. Each ribosome has two parts, a large and a small subunit, as shown
in Figure 1.22. The subunits are attached to each other. Ribosomes can be found alone or in groups within
the cytoplasm. Some ribosomes are attached to the endoplasmic reticulum (as shown in Figure 1.21), and
others are attached to the nuclear envelope.
Ribozymes are RNA molecules that catalyzes chemical reactions, such as translation. Translation is the
process of ordering the amino acids in the assembly of a protein, and more will be discussed on translation
in a later chapter. Briefly, the ribosomes interact with other RNA molecules to make chains of amino acids
called polypeptide chains, due to the peptide bond that forms between individual amino acids. Polypeptide
chains are built from the genetic instructions held within a messenger RNA molecule. Polypeptide chains
that are made on the rough ER are inserted directly into the ER and then are transported to their various
cellular destinations. Ribosomes on the rough ER usually produce proteins that are destined for the cell
membrane.

Figure 1.22: The two subunits that make up a ribosome, small organelles that are intercellular protein
factories.

Golgi Apparatus

The Golgi apparatus is a large organelle that is usually made up of five to eight cup-shaped, membrane-
covered discs called cisternae, as shown in Figure 1.21. The cisternae look a bit like a stack of deflated
balloons. The Golgi apparatus modifies, sorts, and packages different substances for secretion out of the cell,
or for use within the cell. The Golgi apparatus is found close to the nucleus of the cell where it modifies
proteins that have been delivered in transport vesicles from the RER. It is also involved in the transport
of lipids around the cell. Pieces of the Golgi membrane pinch off to form vesicles that transport molecules
around the cell. The Golgi apparatus can be thought of as similar to a post office; it packages and labels
”items” and then sends them to different parts of the cell. Both plant and animal cells have a Golgi apparatus.

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Plant cells can have up to several hundred Golgi stacks scattered throughout the cytoplasm. In plants, the
Golgi apparatus contains enzymes that synthesize some of the cell wall polysaccharides.

Vesicles

A vesicle is a small, spherical compartment that is separated from the cytosol by at least one lipid bilayer.
Many vesicles are made in the Golgi apparatus and the endoplasmic reticulum, or are made from parts of the
cell membrane. Vesicles from the Golgi apparatus can be seen in Figure 1.21. Because it is separated from
the cytosol, the space inside the vesicle can be made to be chemically different from the cytosol. Vesicles are
basic tools of the cell for organizing metabolism, transport, and storage of molecules. Vesicles are also used
as chemical reaction chambers. They can be classified by their contents and function.

• Transport vesicles are able to move molecules between locations inside the cell. For example, trans-
port vesicles move proteins from the rough endoplasmic reticulum to the Golgi apparatus.
• Lysosomes are vesicles that are formed by the Golgi apparatus. They contain powerful enzymes that
could break down (digest) the cell. Lysosomes break down harmful cell products, waste materials,
and cellular debris and then force them out of the cell. They also digest invading organisms such as
bacteria. Lysosomes also break down cells that are ready to die, a process called autolysis.
• Peroxisomes are vesicles that use oxygen to break down toxic substances in the cell. Unlike lysosomes,
which are formed by the Golgi apparatus, peroxisomes self replicate by growing bigger and then divid-
ing. They are common in liver and kidney cells that break down harmful substances. Peroxisomes are
named for the hydrogen peroxide (H2 O2 ) that is produced when they break down organic compounds.
Hydrogen peroxide is toxic, and in turn is broken down into water (H2 O) and oxygen (O2 ) molecules.

Vacuoles

Vacuoles are membrane-bound organelles that can have secretory, excretory, and storage functions. Many
organisms will use vacuoles as storage areas and some plant cells have very large vacuoles. Vesicles are much
smaller than vacuoles and function in transporting materials both within and to the outside of the cell.

Special Structures in Plant Cells

Most of the organelles that have been discussed are common to both animal and plant cells. However, plant
cells also have features that animal cells do not have; they have a cell wall, a large central vacuole, and
plastids such as chloroplasts.
Plants have very different lifestyles from animals, and these differences are apparent when you examine the
structure of the plant cell. Plants make their own food in a process called photosynthesis. They take in
carbon dioxide (CO2 ) and water (H2 O) and convert them into sugars. The features unique to plant cells can
be seen in Figure 1.23.

Cell Wall

A cell wall is a rigid layer that is found outside the cell membrane and surrounds the cell. The cell wall
contains not only cellulose and protein, but other polysaccharides as well. In fact, two other classes of

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Figure 1.23: In addition to containing most of the organelles found in animal cells, plant cells also have a
cell wall, a large central vacuole, and plastids. These three features are not found in animal cells.

polysaccharides, hemicelluloses and pectic polysaccharides, can comprise 30% of the dry mass of the cell
wall. The cell wall provides structural support and protection. Pores in the cell wall allow water and
nutrients to move into and out of the cell. The cell wall also prevents the plant cell from bursting when
water enters the cell.
Microtubules guide the formation of the plant cell wall. Cellulose is laid down by enzymes to form the
primary cell wall. Some plants also have a secondary cell wall. The secondary wall contains a lignin, a
secondary cell component in plant cells that have completed cell growth/expansion.

Central Vacuole

Most mature plant cells have a central vacuole that occupies more than 30% of the cell’s volume, but can
also occupy as much as 90% of the volume of certain cells. The central vacuole is surrounded by a membrane
called the tonoplast. The central vacuole has many functions. Aside from storage, the main role of the
vacuole is to maintain turgor pressure against the cell wall. Proteins found in the tonoplast control the flow
of water into and out of the vacuole. The central vacuole also stores the pigments that color flowers.
The central vacuole contains large amounts of a liquid called cell sap, which differs in composition to the cell
cytosol. Cell sap is a mixture of water, enzymes, ions, salts, and other substances. Cell sap may also contain
toxic byproducts that have been removed from the cytosol. Toxins in the vacuole may help to protect some
plants from being eaten.

Plastids

Plant plastids are a group of closely related membrane-bound organelles that carry out many functions.
They are responsible for photosynthesis, for storage of products such as starch, and for the synthesis of
many types of molecules that are needed as cellular building blocks. Plastids have the ability to change
their function between these and other forms. Plastids contain their own DNA and some ribosomes, and

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scientists think that plastids are descended from photosynthetic bacteria that allowed the first eukaryotes
to make oxygen. The main types of plastids and their functions are:

• Chloroplasts are the organelle of photosynthesis. They capture light energy from the sun and use it
with water and carbon dioxide to make food (sugar) for the plant. The arrangement of chloroplasts in
a plant’s cells can be seen in Figure 1.24.

• Chromoplasts make and store pigments that give petals and fruit their orange and yellow colors.

• Leucoplasts do not contain pigments and are located in roots and non-photosynthetic tissues of
plants. They may become specialized for bulk storage of starch, lipid, or protein. However, in many
cells, leucoplasts do not have a major storage function; instead they make molecules such as fatty acids
and many amino acids.

Figure 1.24: Plant cells with visible chloroplasts (left). Starch-storing potato leucoplasts (right).

Figure 1.25: The internal structure of a chloroplast, with a granal stack of thylakoids circled.

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Chloroplasts capture light energy from the sun and use it with water and carbon dioxide to produce sugars
for food. Chloroplasts look like flat discs that are usually 2 to 10 micrometers in diameter and 1 micrometer
thick. A model of a chloroplast is shown in Figure 1.25. The chloroplast is enclosed by an inner and an
outer phospholipid membrane. Between these two layers is the intermembrane space. The fluid within the
chloroplast is called the stroma, and it contains one or more molecules of small circular DNA. The stroma
also has ribosomes. Within the stroma are stacks of thylakoids, the sub-organelles which are the site of
photosynthesis. The thylakoids are arranged in stacks called grana (singular: granum). A thylakoid has a
flattened disk shape. Inside it is an empty area called the thylakoid space or lumen. Photosynthesis takes
place on the thylakoid membrane.
Within the thylakoid membrane is the complex of proteins and light-absorbing pigments, such as chlorophyll
and carotenoids. This complex allows capture of light energy from many wavelengths because chlorophyll
and carotenoids both absorb different wavelengths of light. You will learn more about how chloroplasts
convert light energy into chemical energy in the Photosynthesis chapter.

Organization of Cells

Biological organization exists at all levels in organisms. It can be seen at the smallest level, in the molecules
that made up such things as DNA and proteins, to the largest level, in an organism such as a blue whale, the
largest mammal on Earth. Similarly, single celled prokaryotes and eukaryotes show order in the way their
cells are arranged. Single-celled organisms such as an amoeba are free-floating and independent-living. Their
single-celled ”bodies” are able to carry out all the processes of life such as metabolism and respiration without
help from other cells. Some single-celled organisms such as bacteria can group together and form a biofilm.
A biofilm is a large grouping of many bacteria that sticks to a surface and makes a protective coating over
itself. Biofilms can show similarities to multicellular organisms. Division of labor is the process in which one
group of cells does one job (such as making the ”glue” that sticks the biofilm to the surface) while another
group of cells does another job (such as taking in nutrients). Multicellular organisms carry out their life
processes through division of labor and they have specialized cells that do specific jobs. However, biofilms are
not considered a multicellular organism and are instead called colonial organisms. The difference between a
multicellular organism and a colonial organism is that individual organisms from a colony or biofilm can, if
separated, survive on their own, while cells from a multicellular organism (e.g., liver cells) cannot.

Colonial Organisms

Colonial organisms were probably one of the first evolutionary steps towards multicellular organisms. Algae
of the genus Volvox are an example of the border between colonial organisms and multicellular organisms.
Each Volvox, shown in Figure 1.26, is a colonial organism. It is made up of between 1000 to 3000 photo-
synthetic algae that are grouped together into a hollow sphere. The sphere has a distinct front and back
end. The cells have eyespots, which are more developed in the cells near the front. This enables the colony
to swim towards light.

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 Figure 1.26: Colonial algae of the genus .

Origin of Multicellularity

The oldest known multicellular organism is a red algae Bangiomorpha pubescens, fossils of which were found
in 1.2 billion year old rock. However, the first organisms were single celled. How multicellular organisms
developed is the subject of much debate.
Scientists think that multicellularity arose from cooperation between many organisms of the same species.
The Colonial Theory proposes that this cooperation led to the development of a multicellular organism.
Many examples of cooperation between organisms in nature have been observed. For example, a certain
species of amoeba (a single-celled animal) groups together during times of food shortage and forms a colony
that moves as one to a new location. Some of these amoebas then become slightly differentiated from each
other. Volvox, shown in Figure 1.26, is another example of a colonial organism. Most scientists accept that
the Colonial theory explains how multicellular organisms evolved.
Multicellular organisms are organisms that are made up of more than one type of cell and have specialized
cells that are grouped together to carry out specialized functions. Most life that you can see without a
microscope is multicellular. As discussed earlier, the cells of a multicellular organism would not survive as
independent cells. The body of a multicellular organism, such as a tree or a cat, exhibits organization at
several levels: tissues, organs, and organ systems. Similar cells are grouped into tissues, groups of tissues
make up organs, and organs with a similar function are grouped into an organ system.

Levels of Organization in Multicellular Organisms

The simplest living multicellular organisms, sponges, are made of many specialized types of cells that work
together for a common goal. Such cell types include digestive cells, tubular pore cells; and epidermal cells.
Though the different cell types create a large organized, multicellular structure—the visible sponge—they
are not organized into true interconnected tissues. If a sponge is broken up by passing it through a sieve,
the sponge will reform on the other side. However, if the sponge’s cells are separated from each other, the
individual cell types cannot survive alone. Simpler colonial organisms, such as members of the genus Volvox,
as shown in Figure 1.26, differ in that their individual cells are free-living and can survive on their own if
separated from the colony.

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Figure 1.27: This roundworm, a multicellular organism, was stained to highlight the nuclei of all the cells in
its body (red dots).

A tissue is a group of connected cells that have a similar function within an organism. More complex
organisms such as jellyfish, coral, and sea anemones have a tissue level of organization. For example, jellyfish
have tissues that have separate protective, digestive, and sensory functions.
Even more complex organisms, such as the roundworm shown in Figure 1.27, while also having differentiated
cells and tissues, have an organ level of development. An organ is a group of tissues that has a specific
function or group of functions. Organs can be as primitive as the brain of a flatworm (a group of nerve cells),
as large as the stem of a sequoia (up to 90 meters, or 300 feet, in height), or as complex as a human liver.
The most complex organisms (such as mammals, trees, and flowers) have organ systems. An organ system
is a group of organs that act together to carry out complex related functions, with each organ focusing on a
part of the task. An example is the human digestive system in which the mouth ingests food, the stomach
crushes and liquifies it, the pancreas and gall bladder make and release digestive enzymes, and the intestines
absorb nutrients into the blood.

Lesson Summary

• The plasma membrane is a selectively permeable lipid bilayer that contains mostly lipids and proteins.
These lipids and proteins are involved in many cellular processes.

• The gel-like material within the cell that holds the organelles is called cytoplasm. The cytosol, which
is the watery substance that does not contain organelles, is made up of 80% to 90% water.

• The cytoskeleton has many functions. It helps to maintain cell shape, it holds organelles in place,
and for some cells, it enables cell movement. The cytoskeleton also plays important roles in both the

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 intracellular movement of substances and in cell division. Three main kinds of cytoskeleton fibers are
microtubules, intermediate filaments, and microfilaments.

• Cilia are extensions of the cell membrane that contain microtubules. Although both are used for
movement, cilia are much shorter than flagella. Cilia cover the surface of some single-celled animals,
such as paramecium, but cover only one side of cells in some multicellular organisms.

• There are three features that plant cells have that animal cells do not have: a cell wall, a large central
vacuole, and plastids.

• Mitochondria use energy from organic compounds to make ATP.

• Ribosomes are exported from the nucleolus, where they are made, to the cytoplasm.

• The Golgi apparatus is a large organelle that is usually made up of five to eight cup-shaped, membrane-
covered discs called cisternae. It modifies, sorts, and packages different substances for secretion out of
the cell, or for use within the cell.

• Individual organisms from a colonial organism or biofilm can, if separated, survive on their own, while
cells from a multicellular organism (e.g., liver cells) cannot.

• A tissue is a group of connected cells that have a similar function within an organism. An organ is a
group of tissues that has a specific function or group of functions, and an organ system is a group of
organs that act together to perform complex related functions, with each organ focusing on a part of
the task.

Review Questions
1. What are the main components of a plasma membrane?

2. What does the fluid mosaic model describe?

3. What is the difference between cytoplasm and cytosol?

4. What type of molecule is common to all three parts of the cytoskeleton?

5. Name the three main parts of the cytoskeleton.

6. What structures do plant cells have that animal cells do not have?

7. Identify two functions of plastids in plant cells.

8. What is the main difference between rough endoplasmic reticulum and smooth endoplasmic reticulum?

9. List five organelles eukaryotes have that prokaryotes do not have.

10. What is a cell feature that distinguishes a colonial organism from a multicellular organism?

11. What is the difference between a cell and a tissue?

12. Identify two functions of the nucleus.

13. Identify the reason why mitochondria are called ”power plants” of the cell.

14. If muscle cells become more active than they usually are, they will grow more mitochondria. Explain
why this happens.

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Further Reading / Supplemental Links
• N. J. Butterfield (2000). Bangiomorpha pubescens n. gen., n. sp.: implications for the evolution of
sex, multicellularity, and the Mesoproterozoic/Neoproterozoic radiation of eukaryotes. Paleobiology 26
(3): 386–404.

• The Bacterial Cytoskeleton. Shih YL, Rothfield L. Microbiol Mol Biol Rev. 2006 Sep;70(3):729-54.

• http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=169

• http://en.wikipedia.orghttp://en.wikipedia.org

Vocabulary
chloroplast The organelle of photosynthesis; captures light energy from the sun and uses it with water
and carbon dioxide to make food (sugar) for the plant.

cilia (cilium) Made up of extensions of the cell membrane that contain microtubules; involved in move-
ment.

cell wall A rigid layer that is found outside the cell membrane and surrounds the cell; provides structural
support and protection.

cytoplasm The gel-like material within the cell that holds the organelles.

cytoskeleton A cellular ”scaffolding” or ”skeleton” that crisscrosses the cytoplasm; helps to maintain cell
shape, it holds organelles in place, and for some cells, it enables cell movement.

endoplasmic reticulum (ER) A network of phospholipid membranes that form hollow tubes, flattened
sheets, and round sacs; involved in transport of molecules, such as proteins, and the synthesis of
proteins and lipids.

flagella (flagellum) Long, thin structures that stick out from the cell membrane; help single-celled or-
ganisms move or swim towards food.

Fluid Mosaic Model Model of the structure of cell membranes; proposes that integral membrane proteins
are embedded in the phospholipid bilayer; some of these proteins extend all the way through the bilayer,
and some only partially across it; also proposes that the membrane behaves like a fluid, rather than a
solid.

gene A short segment of DNA that contains information to encode an RNA molecule or a protein strand.

gene expression The process by which the information in a gene is ”decoded” by various cell molecules
to produce a functional gene product, such as a protein molecule or an RNA molecule.

Golgi apparatus A large organelle that is usually made up of five to eight cup-shaped, membrane-covered
discs called cisternae; modifies, sorts, and packages different substances for secretion out of the cell, or
for use within the cell.

37 www.ck12.org
integral membrane proteins Proteins that are permanently embedded within the plasma membrane;
involved in channeling or transporting molecules across the membrane or acting as cell receptors.

intermediate filaments Filaments that organize the inside structure of the cell by holding organelles and
providing strength.

lipid bilayer A double layer of closely-packed lipid molecules; the cell membrane is a phospholipid bilayer.

lysosome A vesicle that contains powerful digestive enzymes.

membrane protein A protein molecule that is attached to, or associated with the membrane of a cell or
an organelle.

microfilament Filament made of two thin actin chains that are twisted around one another; organizes cell
shape; positions organelles in cytoplasm; involved in cell-to-cell and cell-to-matrix junctions.

microtubules Hollow cylinders that make up the thickest of the cytoskeleton structures; made of the
protein tubulin, with two subunits, alpha and beta tubulin; involved in organelle and vesicle movement;
form mitotic spindles during cell division; involved in cell motility (in cilia and flagella).

mitochondria (mitochondrion) Membrane-enclosed organelles that are found in most eukaryotic cells;
called the ”power plants” of the cell because they use energy from organic compounds to make ATP.

multicellular organisms Organisms that are made up of more than one type of cell; have specialized
cells that are grouped together to carry out specialized functions.

nucleus The membrane-enclosed organelle found in most eukaryotic cells; contains the genetic material
(DNA).

organ A group of tissues that has a specific function or group of functions.

organ system A group of organs that acts together to carry out complex related functions, with each
organ focusing on a part of the task.

peripheral membrane proteins Proteins that are only temporarily associated with the membrane; can
be easily removed, which allows them to be involved in cell signaling.

peroxisomes Vesicles that use oxygen to break down toxic substances in the cell.

phospholipid A lipid made up of up of a polar, phosphorus-containing head, and two long fatty acid,
non-polar ”tails.” The head of the molecule is hydrophilic (water-loving), and the tail is hydrophobic
(water-fearing).

plasma membrane Phospholipid bilayer that separates the internal environment of the cell from the
outside environment.

ribosomes Organelles made of protein and ribosomal RNA (rRNA); where protein synthesis occurs.

www.ck12.org 38
selective permeability The ability to allow only certain molecules in or out of the cell; characteristic of
the cell membrane; also called the cell membrane.

spontaneous generation The belief that living organisms grow directly from decaying organic substances.

tissue A group of connected cells that has a similar function within an organism.

transport vesicle A vesicle that is able to move molecules between locations inside the cell.

vacuole Membrane-bound organelles that can have secretory, excretory, and storage functions; plant cells
have a large central vacuole.

vesicle A small, spherical compartment that is separated from the cytosol by at least one lipid bilayer.

New Points to Consider

• How do you think small molecules, or even water, get through the cell membrane?

• Is it possible that proteins help in this transport process?

• What type of proteins would help with transport?

39 www.ck12.org
© 2015 Authors. This is an open access article published by Portland Press Limited
and distributed under the Creative Commons Attribution License 3.0.
Essays Biochem. (2015) 59, 1–41: doi: 10.1042/BSE0590001

Enzymes: principles and

biotechnological applications
Peter K. Robinson1
College of Science and Technology, University of Central Lancashire, Preston PR1 2HE, U.K.

Abstract
Enzymes are biological catalysts (also known as biocatalysts) that speed up bio-
chemical reactions in living organisms, and which can be extracted from cells and
then used to catalyse a wide range of commercially important processes. This
chapter covers the basic principles of enzymology, such as classification, struc-
ture, kinetics and inhibition, and also provides an overview of industrial applica-
tions. In addition, techniques for the purification of enzymes are discussed.

The nature and classification of enzymes

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions
in living organisms. They can also be extracted from cells and then used to catalyse a wide range of
commercially important processes. For example, they have important roles in the production of
sweetening agents and the modification of antibiotics, they are used in washing powders and vari-
ous cleaning products, and they play a key role in analytical devices and assays that have clinical,
forensic and environmental applications. The word ‘enzyme’ was first used by the German physiol-
ogist Wilhelm Kühne in 1878, when he was describing the ability of yeast to produce alcohol from
sugars, and it is derived from the Greek words en (meaning ‘within’) and zume (meaning ‘yeast’).
In the late nineteenth century and early twentieth century, significant advances were made in
the extraction, characterization and commercial exploitation of many enzymes, but it was not until
the 1920s that enzymes were crystallized, revealing that catalytic activity is associated with protein
molecules. For the next 60 years or so it was believed that all enzymes were proteins, but in the
1To whom correspondence should be addressed (email [email protected]).
This article is a reviewed, revised and updated version of the following ‘Biochemistry Across the School
Curriculum’ (BASC) booklet: Teal A.R. & Wymer P.E.O., 1995: Enzymes and their Role in Technology. For further
information and to provide feedback on this or any other Biochemical Society education resource, please contact
[email protected]. For further information on other Biochemical Society publications, please visit www.
biochemistry.org/publications.

1
2 Essays in Biochemistry volume 59 2015

1980s it was found that some ribonucleic acid (RNA) molecules are also able to exert catalytic
effects. These RNAs, which are called ribozymes, play an important role in gene expression. In the
same decade, biochemists also developed the technology to generate antibodies that possess cata-
lytic properties. These so-called ‘abzymes’ have significant potential both as novel industrial cata-
lysts and in therapeutics. Notwithstanding these notable exceptions, much of classical enzymology,
and the remainder of this essay, is focused on the proteins that possess catalytic activity.
As catalysts, enzymes are only required in very low concentrations, and they speed up
reactions without themselves being consumed during the reaction. We usually describe
enzymes as being capable of catalysing the conversion of substrate molecules into product
molecules as follows:
Enzyme

Substrate  
 Product


Enzymes are potent catalysts

The enormous catalytic activity of enzymes can perhaps best be expressed by a constant, kcat, that
is variously referred to as the turnover rate, turnover frequency or turnover number. This con-
stant represents the number of substrate molecules that can be converted to product by a single
enzyme molecule per unit time (usually per minute or per second). Examples of turnover rate
values are listed in Table 1. For example, a single molecule of carbonic anhydrase can catalyse the
conversion of over half a million molecules of its substrates, carbon dioxide (CO2) and water
(H2O), into the product, bicarbonate (HCO3−), every second—a truly remarkable achievement.

Enzymes are specific catalysts

As well as being highly potent catalysts, enzymes also possess remarkable specificity in that
they generally catalyse the conversion of only one type (or at most a range of similar types) of
substrate molecule into product molecules.
Some enzymes demonstrate group specificity. For example, alkaline phosphatase (an
enzyme that is commonly encountered in first-year laboratory sessions on enzyme kinetics)
can remove a phosphate group from a variety of substrates.
Other enzymes demonstrate much higher specificity, which is described as absolute speci-
ficity. For example, glucose oxidase shows almost total specificity for its substrate, β-D-glucose,
and virtually no activity with any other monosaccharides. As we shall see later, this specificity
is of paramount importance in many analytical assays and devices (biosensors) that measure a
specific substrate (e.g. glucose) in a complex mixture (e.g. a blood or urine sample).

Table 1. Turnover rate of some common enzymes showing wide variation.

Enzyme Turnover rate (mole product s−1 mole enzyme−1)

Carbonic anhydrase 600 000

Catalase 93 000

β–galactosidase 200

Chymotrypsin 100

Tyrosinase 1

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P.K. Robinson 3

Enzyme names and classification

Enzymes typically have common names (often called ‘trivial names’) which refer to the reaction
that they catalyse, with the suffix -ase (e.g. oxidase, dehydrogenase, carboxylase), although individ-
ual proteolytic enzymes generally have the suffix -in (e.g. trypsin, chymotrypsin, papain). Often
the trivial name also indicates the substrate on which the enzyme acts (e.g. glucose oxidase, alco-
hol dehydrogenase, pyruvate decarboxylase). However, some trivial names (e.g. invertase, diastase,
catalase) provide little information about the substrate, the product or the reaction involved.
Due to the growing complexity of and inconsistency in the naming of enzymes, the
International Union of Biochemistry set up the Enzyme Commission to address this issue. The first
Enzyme Commission Report was published in 1961, and provided a systematic approach to the
naming of enzymes. The sixth edition, published in 1992, contained details of nearly 3 200 different
enzymes, and supplements published annually have now extended this number to over 5 000.
Within this system, all enzymes are described by a four-part Enzyme Commission (EC)
number. For example, the enzyme with the trivial name lactate dehydrogenase has the EC
number 1.1.1.27, and is more correctly called l–lactate: NAD+ oxidoreductase.
The first part of the EC number refers to the reaction that the enzyme catalyses (Table 2).
The remaining digits have different meanings according to the nature of the reaction identified
by the first digit. For example, within the oxidoreductase category, the second digit denotes the
hydrogen donor (Table 3) and the third digit denotes the hydrogen acceptor (Table 4).
Thus lactate dehydrogenase with the EC number 1.1.1.27 is an oxidoreductase (indicated
by the first digit) with the alcohol group of the lactate molecule as the hydrogen donor (second
digit) and NAD+ as the hydrogen acceptor (third digit), and is the 27th enzyme to be catego-
rized within this group (fourth digit).

Table 2. Enzyme Classification: Main classes of enzymes in EC system.

First EC digit Enzyme class Reaction type

1. Oxidoreductases Oxidation/reduction

2. Transferases Atom/group transfer (excluding other classes)

3. Hydrolases Hydrolysis

4. Lyases Group removal (excluding 3.)

5. Isomerases Isomerization

6. Ligases Joining of molecules linked to the breakage of

a pyrophosphate bond

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4 Essays in Biochemistry volume 59 2015

Table 3. Enzyme Classification: Secondary classes of oxidoreductase enzymes

in EC system.

Oxidoreductases: Hydrogen or electron donor

second EC digit
1. Alcohol (CHOH)

2. Aldehyde or ketone (C═O)

3. ─CH─CH─

4. Primary amine (CHNH2 or CHNH3+)

5. Secondary amine (CHNH)

6. NADH or NADPH (when another redox catalyst is the acceptor)

Table 4. Enzyme Classification: Tertiary classes of oxidoreductase enzymes in

EC system.

Oxidoreductases: third EC digit Hydrogen or electron acceptor

1. NAD+ or NADP+

2. Fe3+ (e.g. cytochromes)

3. O2

4. Other

Fortunately, it is now very easy to find this information for any individual enzyme using
the Enzyme Nomenclature Database (available at http://enzyme.expasy.org).

Enzyme structure and substrate binding

Amino acid-based enzymes are globular proteins that range in size from less than 100 to more
than 2 000 amino acid residues. These amino acids can be arranged as one or more polypep-
tide chains that are folded and bent to form a specific three-dimensional structure, incorporat-
ing a small area known as the active site (Figure 1), where the substrate actually binds. The
active site may well involve only a small number (less than 10) of the constituent amino acids.
It is the shape and charge properties of the active site that enable it to bind to a single type
of substrate molecule, so that the enzyme is able to demonstrate considerable specificity in its
catalytic activity.
The hypothesis that enzyme specificity results from the complementary nature of the sub-
strate and its active site was first proposed by the German chemist Emil Fischer in 1894, and
became known as Fischer’s ‘lock and key hypothesis’, whereby only a key of the correct size and
shape (the substrate) fits into the keyhole (the active site) of the lock (the enzyme). It is
astounding that this theory was proposed at a time when it was not even established that

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P.K. Robinson 5

Figure 1. Representation of substrate binding to the active site of an enzyme

molecule.

enzymes were proteins. As more was learned about enzyme structure through techniques such
as X-ray crystallography, it became clear that enzymes are not rigid structures, but are in fact
quite flexible in shape. In the light of this finding, in 1958 Daniel Koshland extended Fischer’s
ideas and presented the ‘induced-fit model’ of substrate and enzyme binding, in which the
enzyme molecule changes its shape slightly to accommodate the binding of the substrate. The
analogy that is commonly used is the ‘hand-in-glove model’, where the hand and glove are
broadly complementary in shape, but the glove is moulded around the hand as it is inserted in
order to provide a perfect match.
Since it is the active site alone that binds to the substrate, it is logical to ask what is the
role of the rest of the protein molecule. The simple answer is that it acts to stabilize the
active site and provide an appropriate environment for interaction of the site with the sub-
strate molecule. Therefore the active site cannot be separated out from the rest of the protein
without loss of catalytic activity, although laboratory-based directed (or forced) evolution
studies have shown that it is sometimes possible to generate smaller enzymes that do retain
activity.
It should be noted that although a large number of enzymes consist solely of protein,
many also contain a non-protein component, known as a cofactor, that is necessary for the
enzyme’s catalytic activity. A cofactor may be another organic molecule, in which case it is
called a coenzyme, or it may be an inorganic molecule, typically a metal ion such as iron, man-
ganese, cobalt, copper or zinc. A coenzyme that binds tightly and permanently to the protein is
generally referred to as the prosthetic group of the enzyme.
When an enzyme requires a cofactor for its activity, the inactive protein component is
generally referred to as an apoenzyme, and the apoenzyme plus the cofactor (i.e. the active
enzyme) is called a holoenzyme (Figure 2).
The need for minerals and vitamins in the human diet is partly attributable to their roles
within metabolism as cofactors and coenzymes.

Enzymes and reaction equilibrium

How do enzymes work? The broad answer to this question is that they do not alter the equilib-
rium (i.e. the thermodynamics) of a reaction. This is because enzymes do not fundamentally
change the structure and energetics of the products and reagents, but rather they simply allow

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6 Essays in Biochemistry volume 59 2015

Figure 2. The components of a holoenzyme.

the reaction equilibrium to be attained more rapidly. Let us therefore begin by clarifying the
concept of chemical equilibrium.
In many cases the equilibrium of a reaction is far ‘to the right’—that is, virtually all of the
substrate (S) is converted into product (P). For this reason, reactions are often written as
follows:

S→P
This is a simplification, as in all cases it is more correct to write this reaction as follows:

SP
This indicates the presence of an equilibrium. To understand this concept it is perhaps
most helpful to look at a reaction where the equilibrium point is quite central.
For example:

Glucose isomerase

Glucose 
 Fructose

In this reaction, if we start with a solution of 1 mol l−1 glucose and add the enzyme, then
upon completion we will have a mixture of approximately 0.5 mol l−1 glucose and 0.5 mol l−1
fructose. This is the equilibrium point of this particular reaction, and although it may only
take a couple of seconds to reach this end point with the enzyme present, we would in fact
come to the same point if we put glucose into solution and waited many months for the reac-
tion to occur in the absence of the enzyme. Interestingly, we could also have started this reac-
tion with a 1 mol l−1 fructose solution, and it would have proceeded in the opposite direction
until the same equilibrium point had been reached.
The equilibrium point for this reaction is expressed by the equilibrium constant Keq as
follows:

Substrate concentration at end point 0.5

K eq = = =1
Substrate concentration at end point 0.5

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P.K. Robinson 7

Thus for a reaction with central equilibrium, Keq = 1, for an equilibrium ‘to the right’ Keq
is >1, and for an equilibrium ‘to the left’ Keq is <1.
Therefore if a reaction has a Keq value of 106, the equilibrium is very far to the right and
can be simplified by denoting it as a single arrow. We may often describe this type of reaction
as ‘going to completion’. Conversely, if a reaction has a Keq value of 10−6, the equilibrium is very
far to the left, and for all practical purposes it would not really be considered to proceed at all.
It should be noted that although the concentration of reactants has no effect on the equi-
librium point, environmental factors such as pH and temperature can and do affect the posi-
tion of the equilibrium.
It should also be noted that any biochemical reaction which occurs in vivo in a living sys-
tem does not occur in isolation, but as part of a metabolic pathway, which makes it more diffi-
cult to conceptualize the relationship between reactants and reactions. In vivo reactions are not
allowed to proceed to their equilibrium position. If they did, the reaction would essentially
stop (i.e. the forward and reverse reactions would balance each other), and there would be no
net flux through the pathway. However, in many complex biochemical pathways some of the
individual reaction steps are close to equilibrium, whereas others are far from equilibrium, the
latter (catalysed by regulatory enzymes) having the greatest capacity to control the overall flux
of materials through the pathway.

Enzymes form complexes with their substrates

We often describe an enzyme-catalysed reaction as proceeding through three stages as follows:

E + S → ES complex → E + P

The ES complex represents a position where the substrate (S) is bound to the enzyme (E)
such that the reaction (whatever it might be) is made more favourable. As soon as the reaction
has occurred, the product molecule (P) dissociates from the enzyme, which is then free to bind
to another substrate molecule. At some point during this process the substrate is converted
into an intermediate form (often called the transition state) and then into the product.
The exact mechanism whereby the enzyme acts to increase the rate of the reaction differs
from one system to another. However, the general principle is that by binding of the substrate
to the enzyme, the reaction involving the substrate is made more favourable by lowering the
activation energy of the reaction.
In terms of energetics, reactions can be either exergonic (releasing energy) or endergonic
(consuming energy). However, even in an exergonic reaction a small amount of energy,
termed the activation energy, is needed to give the reaction a ‘kick start.’ A good analogy is that
of a match, the head of which contains a mixture of energy-rich chemicals (phosphorus ses-
quisulfide and potassium chlorate). When a match burns it releases substantial amounts of
light and heat energy (exergonically reacting with O2 in the air). However, and perhaps fortu-
nately, a match will not spontaneously ignite, but rather a small input of energy in the form of
heat generated through friction (i.e. striking of the match) is needed to initiate the reaction. Of
course once the match has been struck the amount of energy released is considerable, and
greatly exceeds the small energy input during the striking process.
As shown in Figure 3, enzymes are considered to lower the activation energy of a system
by making it energetically easier for the transition state to form. In the presence of an enzyme

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8 Essays in Biochemistry volume 59 2015

Figure 3. Effect of an enzyme on reducing the activation energy required to start a

reaction where (a) is uncatalysed and (b) is enzyme-catalysed reaction.

catalyst, the formation of the transition state is energetically more favourable (i.e. it requires
less energy for the ‘kick start’), thereby accelerating the rate at which the reaction will proceed,
but not fundamentally changing the energy levels of either the reactant or the product.

Properties and mechanisms of enzyme

action
Enzyme kinetics
Enzyme kinetics is the study of factors that determine the speed of enzyme-catalysed reac-
tions. It utilizes some mathematical equations that can be confusing to students when they first
encounter them. However, the theory of kinetics is both logical and simple, and it is essential
to develop an understanding of this subject in order to be able to appreciate the role of
enzymes both in metabolism and in biotechnology.
Assays (measurements) of enzyme activity can be performed in either a discontinuous or
continuous fashion. Discontinuous methods involve mixing the substrate and enzyme together
and measuring the product formed after a set period of time, so these methods are generally easy
and quick to perform. In general we would use such discontinuous assays when we know little
about the system (and are making preliminary investigations), or alternatively when we know a
great deal about the system and are certain that the time interval we are choosing is appropriate.
In continuous enzyme assays we would generally study the rate of an enzyme-catalysed
reaction by mixing the enzyme with the substrate and continuously measuring the appear-
ance of product over time. Of course we could equally well measure the rate of the reaction
by measuring the disappearance of substrate over time. Apart from the actual direction (one
increasing and one decreasing), the two values would be identical. In enzyme kinetics experi-
ments, for convenience we very often use an artificial substrate called a chromogen that yields

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P.K. Robinson 9

a brightly coloured product, making the reaction easy to follow using a colorimeter or a spec-
trophotometer. However, we could in fact use any available analytical equipment that has the
capacity to measure the concentration of either the product or the substrate.

In almost all cases we would also add a buffer solution to the mixture. As we shall see,
enzyme activity is strongly influenced by pH, so it is important to set the pH at a specific value
and keep it constant throughout the experiment.
Our first enzyme kinetics experiment may therefore involve mixing a substrate solution
(chromogen) with a buffer solution and adding the enzyme. This mixture would then be
placed in a spectrophotometer and the appearance of the coloured product would be meas-
ured. This would enable us to follow a rapid reaction which, after a few seconds or minutes,
might start to slow down, as shown in Figure 4.
A common reason for this slowing down of the speed (rate) of the reaction is that the
substrate within the mixture is being used up and thus becoming limiting. Alternatively, it may
be that the enzyme is unstable and is denaturing over the course of the experiment, or it could
be that the pH of the mixture is changing, as many reactions either consume or release pro-
tons. For these reasons, when we are asked to specify the rate of a reaction we do so early on,
as soon as the enzyme has been added, and when none of the above-mentioned limitations

Figure 4. Formation of product in an enzyme-catalysed reaction, plotted against time.

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10 Essays in Biochemistry volume 59 2015

apply. We refer to this initial rapid rate as the initial velocity (v0). Measurement of the reaction
rate at this early stage is also quite straightforward, as the rate is effectively linear, so we can
simply draw a straight line and measure the gradient (by dividing the concentration change by
the time interval) in order to evaluate the reaction rate over this period.
We may now perform a range of similar enzyme assays to evaluate how the initial velocity
changes when the substrate or enzyme concentration is altered, or when the pH is changed.
These studies will help us to characterize the properties of the enzyme under study.
The relationship between enzyme concentration and the rate of the reaction is usually a
simple one. If we repeat the experiment just described, but add 10% more enzyme, the reaction
will be 10% faster, and if we double the enzyme concentration the reaction will proceed twice
as fast. Thus there is a simple linear relationship between the reaction rate and the amount of
enzyme available to catalyse the reaction (Figure 5).
This relationship applies both to enzymes in vivo and to those used in biotechnologi-
cal applications, where regulation of the amount of enzyme present may control reaction
rates.
When we perform a series of enzyme assays using the same enzyme concentration,
but with a range of different substrate concentrations, a slightly more complex relation-
ship emerges, as shown in Figure 6. Initially, when the substrate concentration is
increased, the rate of reaction increases considerably. However, as the substrate concentra-
tion is increased further the effects on the reaction rate start to decline, until a stage is
reached where increasing the substrate concentration has little further effect on the reac-
tion rate. At this point the enzyme is considered to be coming close to saturation with
substrate, and demonstrating its maximal velocity (Vmax). Note that this maximal velocity
is in fact a theoretical limit that will not be truly achieved in any experiment, although we
might come very close to it.

Figure 5. Relationship between enzyme concentration and the rate of an enzyme-

catalysed reaction.

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P.K. Robinson 11

Figure 6. Relationship between substrate concentration and the rate of an enzyme-

catalysed reaction.

The relationship described here is a fairly common one, which a mathematician would
immediately identify as a rectangular hyperbola. The equation that describes such a relation-
ship is as follows:
a×x
y=
x+b

The two constants a and b thus allow us to describe this hyperbolic relationship, just as
with a linear relationship (y = mx + c), which can be expressed by the two constants m (the
slope) and c (the intercept).
We have in fact already defined the constant a — it is Vmax. The constant b is a little more
complex, as it is the value on the x-axis that gives half of the maximal value of y. In enzymol-
ogy we refer to this as the Michaelis constant (Km), which is defined as the substrate concentra-
tion that gives half-maximal velocity.
Our final equation, usually called the Michaelis–Menten equation, therefore becomes:

Vmax × Substrate concentration

Initial rate of reaction (v0 )=
Substrate concentration + K m

In 1913, Leonor Michaelis and Maud Menten first showed that it was in fact possible to
derive this equation mathematically from first principles, with some simple assumptions about
the way in which an enzyme reacts with a substrate to form a product. Central to their deriva-
tion is the concept that the reaction takes place via the formation of an ES complex which,
once formed, can either dissociate (productively) to release product, or else dissociate in the
reverse direction without any formation of product. Thus the reaction can be represented as
follows, with k1, k−1 and k2 being the rate constants of the three individual reaction steps:
k1

E + S  ES k
2
→E + P
k−1 

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12 Essays in Biochemistry volume 59 2015

The Michaelis–Menten derivation requires two important assumptions. The first assump-
tion is that we are considering the initial velocity of the reaction (v0), when the product con-
centration will be negligibly small (i.e. [S] ≫ [P]), such that we can ignore the possibility of
any product reverting to substrate. The second assumption is that the concentration of sub-
strate greatly exceeds the concentration of enzyme (i.e. [S] ≫ [E]).
The derivation begins with an equation for the expression of the initial rate, the rate
of formation of product, as the rate at which the ES complex dissociates to form product.
This is based upon the rate constant k 2 and the concentration of the ES complex, as
follows:

d[P]
v0 = = k2 × [ES] (1)
dt

Since ES is an intermediate, its concentration is unknown, but we can express it in terms

of known values. In a steady-state approximation we can assume that although the concentra-
tion of substrate and product changes, the concentration of the ES complex itself remains con-
stant. The rate of formation of the ES complex and the rate of its breakdown must therefore
balance, where:

Rate of ES complex formation = k1[E][S]

and

Rate of ES complex breakdown = (k−1 + k2 )[ES]

Hence, at steady state:

k1[E][S] = k−1 + k2[ES]

This equation can be rearranged to yield [ES] as follows:

k1[E][S]
[ES] = (2)
k−1 + k2

The Michaelis constant Km can be defined as follows:

k−1 + k2
Km =
k1

Equation 2 may thus be simplified to:

[E][S]
[ES] = (3)
Km
Since the concentration of substrate greatly exceeds the concentration of enzyme (i.e.
[S] ≫ [E]), the concentration of uncombined substrate [S] is almost equal to the total concen-
tration of substrate. The concentration of uncombined enzyme [E] is equal to the total enzyme

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P.K. Robinson 13

concentration [E]T minus that combined with substrate [ES]. Introducing these terms to
Equation 3 and solving for ES gives us the following:

[E]T[S]
[ES] = (4)
[S] + K m

We can then introduce this term into Equation 1 to give:

[S]
v0 = k2[E]T (5)
[S] + K m

The term k2[E]T in fact represents Vmax, the maximal velocity. Thus Michaelis and Menten
were able to derive their final equation as:

Vmax [S]
v0 =
[S] + K m

A more detailed derivation of the Michaelis–Menten equation can be found in many bio-
chemistry textbooks (see section 4 of Recommended Reading section). There are also some very
helpful web-based tutorials available on the subject.
Michaelis constants have been determined for many commonly used enzymes, and are
typically in the lower millimolar range (Table 5).
It should be noted that enzymes which catalyse the same reaction, but which are derived
from different organisms, can have widely differing Km values. Furthermore, an enzyme with
multiple substrates can have quite different Km values for each substrate.
A low Km value indicates that the enzyme requires only a small amount of substrate in
order to become saturated. Therefore the maximum velocity is reached at relatively low sub-
strate concentrations. A high Km value indicates the need for high substrate concentrations in
order to achieve maximum reaction velocity. Thus we generally refer to Km as a measure of the
affinity of the enzyme for its substrate—in fact it is an inverse measure, where a high Km indi-
cates a low affinity, and vice versa.
The Km value tells us several important things about a particular enzyme.

Table 5. Typical range of values of the Michaelis constant.

Enzyme Km (mmol l−1)

Carbonic anhydrase 26

Chymotrypsin 15

Ribonuclease 8

Tyrosyl-tRNA synthetase 0.9

Pepsin 0.3

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14 Essays in Biochemistry volume 59 2015

1. An enzyme with a low Km value relative to the physiological concentration of substrate will
probably always be saturated with substrate, and will therefore act at a constant rate,
regardless of variations in the concentration of substrate within the physiological range.
2. An enzyme with a high Km value relative to the physiological concentration of substrate will
not be saturated with substrate, and its activity will therefore vary according to the concen-
tration of substrate, so the rate of formation of product will depend on the availability of
substrate.
3. If an enzyme acts on several substrates, the substrate with the lowest Km value is fre-
quently assumed to be that enzyme’s ‘natural’ substrate, although this may not be true in
all cases.
4. If two enzymes (with similar Vmax) in different metabolic pathways compete for the same
substrate, then if we know the Km values for the two enzymes we can predict the relative
activity of the two pathways. Essentially the pathway that has the enzyme with the lower Km
value is likely to be the ‘preferred pathway’, and more substrate will flow through that path-
way under most conditions. For example, phosphofructokinase (PFK) is the enzyme that
catalyses the first committed step in the glycolytic pathway, which generates energy in the
form of ATP for the cell, whereas glucose-1-phosphate uridylyltransferase (GUT) is an
enzyme early in the pathway leading to the synthesis of glycogen (an energy storage mole-
cule). Both enzymes use hexose monophosphates as substrates, but the Km of PFK for its
substrate is lower than that of GUT for its substrate. Thus at lower cellular hexose phos-
phate concentrations, PFK will be active and GUT will be largely inactive. At higher hexose
phosphate concentrations both pathways will be active. This means that the cells only store
glycogen in times of plenty, and always give preference to the pathway of ATP production,
which is the more essential function.

Very often it is not possible to estimate Km values from a direct plot of velocity against
substrate concentration (as shown in Figure 6) because we have not used high enough

Figure 7. (a) Direct plot. (b) Lineweaver–Burk plot of the same kinetic data.

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P.K. Robinson 15

substrate concentrations to come even close to estimating maximal velocity, and therefore we
cannot evaluate half-maximal velocity and thus Km. Fortunately, we can plot our experimental
data in a slightly different way in order to obtain these values. The most commonly used
alternative is the Lineweaver–Burk plot (often called the double-reciprocal plot). This plot
linearizes the hyperbolic curved relationship, and the line produced is easy to extrapolate,
allowing evaluation of Vmax and Km. For example, if we obtained only the first seven data
points in Figure 6, we would have difficulty estimating Vmax from a direct plot as shown in
Figure 7a.
However, as shown in Figure 7b, if these seven points are plotted on a graph of 1/velocity
against 1/substrate concentration (i.e. a double-reciprocal plot), the data are linearized, and the
line can be easily extrapolated to the left to provide intercepts on both the y-axis and the
x-axis, from which Vmax and Km, respectively, can be evaluated.
One significant practical drawback of using the Lineweaver–Burk plot is the excessive
influence that it gives to measurements made at the lowest substrate concentrations. These
concentrations might well be the most prone to error (due to difficulties in making multiple
dilutions), and result in reaction rates that, because they are slow, might also be most prone to
measurement error. Often, as shown in Figure 8, such points when transformed on the
Lineweaver–Burk plot have a significant impact on the line of best fit estimated from the data,
and therefore on the extrapolated values of both Vmax and Km. The two sets of points shown in
Figure 8 are identical except for the single point at the top right, which reflects (because of the
plot’s double-reciprocal nature) a single point derived from a very low substrate concentration
and a low reaction rate. However, this single point can have an enormous impact on the line of
best fit and the accompanying estimates of kinetic constants.
In fact there are other kinetic plots that can be used, including the Eadie–Hofstee plot,
the Hanes plot and the Eisenthal–Cornish-Bowden plot, which are less prone to such prob-
lems. However, the Lineweaver–Burk plot is still the most commonly described kinetic plot in
the majority of enzymology textbooks, and thus retains its influence in undergraduate
education.

Figure 8. Lineweaver–Burk plot of similar kinetic data, which differ only in a single.
(Final data point (a) 1/v 0.03 at 1/S of 0.2 and (b) 1/v 0.031 at 1/S of 0.18).

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16 Essays in Biochemistry volume 59 2015

Figure 9. The pH profile of β-glucosidase.

Enzymes are affected by pH and temperature

Various environmental factors are able to affect the rate of enzyme-catalysed reactions through
reversible or irreversible changes in the protein structure. The effects of pH and temperature
are generally well understood.
Most enzymes have a characteristic optimum pH at which the velocity of the catalysed
reaction is maximal, and above and below which the velocity declines (Figure 9).
The pH profile is dependent on a number of factors. As the pH changes, the ionization of
groups both at the enzyme’s active site and on the substrate can alter, influencing the rate of
binding of the substrate to the active site. These effects are often reversible. For example, if we
take an enzyme with an optimal pH (pHopt) of 7.0 and place it in an environment at pH 6.0 or
8.0, the charge properties of the enzyme and the substrate may be suboptimal, such that bind-
ing and hence the reaction rate are lowered. If we then readjust the pH to 7.0, the optimal
charge properties and hence the maximal activity of the enzyme are often restored. However, if
we place the enzyme in a more extreme acidic or alkaline environment (e.g. at pH 1 or 14),
although these conditions may not actually lead to changes in the very stable covalent struc-
ture of the protein (i.e. its configuration), they may well produce changes in the conformation
(shape) of the protein such that, when it is returned to pH 7.0, the original conformation and
hence the enzyme’s full catalytic activity are not restored.
It should be noted that the optimum pH of an enzyme may not be identical to that of its
normal intracellular surroundings. This indicates that the local pH can exert a controlling
influence on enzyme activity.
The effects of temperature on enzyme activity are quite complex, and can be regarded as
two forces acting simultaneously but in opposite directions. As the temperature is raised, the
rate of molecular movement and hence the rate of reaction increases, but at the same time
there is a progressive inactivation caused by denaturation of the enzyme protein. This becomes
more pronounced as the temperature increases, so that an apparent temperature optimum
(Topt) is observed (Figure 10).

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Figure 10. The effect of temperature on enzyme activity.

Thermal denaturation is time dependent, and for an enzyme the term ‘optimum tempera-
ture’ has little real meaning unless the duration of exposure to that temperature is recorded.
The thermal stability of an enzyme can be determined by first exposing the protein to a range
of temperatures for a fixed period of time, and subsequently measuring its activity at one
favourable temperature (e.g. 25°C).
The temperature at which denaturation becomes important varies from one enzyme to
another. Normally it is negligible below 30°C, and starts to become appreciable above 40°C.
Typically, enzymes derived from microbial sources show much higher thermal stability than
do those from mammalian sources, and enzymes derived from extremely thermophilic micro-
organisms, such as thermolysin (a protease from Bacillus thermoproteolyticus) and Taq poly-
merase (a DNA polymerase from Thermus aquaticus), might be completely thermostable at
70°C and still retain substantial levels of activity even at 100°C.

Enzymes are sensitive to inhibitors

Substances that reduce the activity of an enzyme-catalysed reaction are known as inhibitors.
They act by either directly or indirectly influencing the catalytic properties of the active site.
Inhibitors can be foreign to the cell or natural components of it. Those in the latter category
can represent an important element of the regulation of cell metabolism. Many toxins and also
many pharmacologically active agents (both illegal drugs and prescription and over-the-
counter medicines) act by inhibiting specific enzyme-catalysed processes.

Reversible inhibition
Inhibitors are classified as reversible inhibitors when they bind reversibly to an enzyme. A
molecule that is structurally similar to the normal substrate may be able to bind reversibly to
the enzyme’s active site and therefore act as a competitive inhibitor. For example, malonate is a
competitive inhibitor of the enzyme succinate dehydrogenase, as it is capable of binding to the
enzyme’s active site due to its close structural similarity to the enzyme’s natural substrate, suc-
cinate (see below). When malonate occupies the active site of succinate dehydrogenase it

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18 Essays in Biochemistry volume 59 2015

prevents the natural substrate, succinate, from binding, thereby slowing down the rate of oxi-
dation of succinate to fumarate (i.e. inhibiting the reaction).

One of the characteristics of competitive inhibitors is that they can be displaced from the
active site if high concentrations of substrate are used, thereby restoring enzyme activity. Thus
competitive inhibitors increase the Km of a reaction because they increase the concentration of
substrate required to saturate the enzyme. However, they do not change Vmax itself.
In the case of certain enzymes, high concentrations of either the substrate or the product
can be inhibitory. For example, invertase activity is considerably reduced in the presence of
high concentrations of sucrose (its substrate), whereas the β-galactosidase of Aspergillus niger
is strongly inhibited by galactose (its product). Products of an enzyme reaction are some of the
most commonly encountered competitive inhibitors.
Other types of reversible inhibitor also exist. Non-competitive inhibitors react with the
enzyme at a site distinct from the active site. Therefore the binding of the inhibitor does not
physically block the substrate–binding site, but it does prevent subsequent reaction. Most non-
competitive inhibitors are chemically unrelated to the substrate, and their inhibition cannot be
overcome by increasing the substrate concentration. Such inhibitors in effect reduce the con-
centration of the active enzyme in solution, thereby reducing the Vmax of the reaction.
However, they do not change the value of Km.
Uncompetitive inhibition is rather rare, occurring when the inhibitor is only able to bind
to the enzyme once a substrate molecule has itself bound. As such, inhibition is most signifi-
cant at high substrate concentrations, and results in a reduction in the Vmax of the reaction.
Uncompetitive inhibition also causes a reduction in Km, which seems somewhat counterintui-
tive as this means that the affinity of the enzyme for its substrate is actually increased when the
inhibitor is present. This effect occurs because the binding of the inhibitor to the ES complex
effectively removes ES complex and thereby affects the overall equilibrium of the reaction
favouring ES complex formation. It is noteworthy however that since both Vmax and Km are

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P.K. Robinson 19

reduced the observed reaction rates with inhibitor present are always lower than those in the
absence of the uncompetitive inhibitor.

Irreversible inhibitors and poisons

If an inhibitor binds permanently to an enzyme it is known as an irreversible inhibitor. Many
irreversible inhibitors are therefore potent toxins.
Organophosphorus compounds such as diisopropyl fluorophosphate (DFP) inhibit ace-
tylcholinesterase activity by reacting covalently with an important serine residue found within
the active site of the enzyme. The physiological effect of this inactivation is interference with
neurotransmitter inactivation at the synapses of nerves, resulting in the constant propagation
of nerve impulses, which can lead to death. DFP was originally evaluated by the British as a
chemical warfare agent during World War Two, and modified versions of this compound are
now widely used as organophosphate pesticides (e.g. parathione, malathione).

Allosteric regulators and the control of enzyme activity

Having spent time learning about enzyme kinetics and the Michaelis–Menten relationship, it is
often quite disconcerting to find that some of the most important enzymes do not in fact display
such properties. Allosteric enzymes are key regulatory enzymes that control the activities of met-
abolic pathways by responding to inhibitors and activators. These enzymes in fact show a sigmoi-
dal (S-shaped) relationship between reaction rate and substrate concentration (Figure 11), rather
than the usual hyperbolic relationship. Thus for allosteric enzymes there is an area where activity
is lower than that of an equivalent ‘normal’ enzyme, and also an area where activity is higher than
that of an equivalent ‘normal’ enzyme, with a rapid transition between these two phases. This is
rather like a switch that can quickly be changed from ‘off ’ (low activity) to ‘on’ (full activity).
Most allosteric enzymes are polymeric—that is, they are composed of at least two (and
often many more) individual polypeptide chains. They also have multiple active sites where the
substrate can bind. Much of our understanding of the function of allosteric enzymes comes

Figure 11. Activity/substrate profiles of allosteric (⚬) and non-allosteric (•) enzymes
with the same affinity and maximal velocity.

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from studies of haemoglobin which, although it is not an enzyme, binds oxygen in a similarly
co-operative way and thus also demonstrates this sigmoidal relationship. Allosteric enzymes
have an initially low affinity for the substrate, but when a single substrate molecule binds, this
may break some bonds within the enzyme and thereby change the shape of the protein such
that the remaining active sites are able to bind with a higher affinity. Therefore allosteric
enzymes are often described as moving from a tensed state or T-state (low affinity) in which no
substrate is bound, to a relaxed state or R-state (high affinity) as substrate binds. Other mole-
cules can also bind to allosteric enzymes, at additional regulatory sites (i.e. not at the active site).
Molecules that stabilize the protein in its T-state therefore act as allosteric inhibitors, whereas
molecules that move the protein to its R-state will act as allosteric activators or promoters.
A good example of an allosteric enzyme is aspartate transcarbamoylase (ATCase), a key
regulatory enzyme that catalyses the first committed step in the sequence of reactions that pro-
duce the pyrimidine nucleotides which are essential components of DNA and RNA. The reac-
tion is as follows:

The end product in the pathway, the pyrimidine nucleotide cytidine triphosphate (CTP), is
an active allosteric inhibitor of the enzyme ATCase. Therefore when there is a high concentra-
tion of CTP in the cell, this feeds back and inhibits the ATCase enzyme, reducing its activity
and thus lowering the rate of production of further pyrimidine nucleotides. As the concentra-
tion of CTP in the cell decreases then so does the inhibition of ATCase, and the resulting
increase in enzyme activity leads to the production of more pyrimidine nucleotides. This nega-
tive feedback inhibition is an important element of biochemical homeostasis within the cell.
However, in order to synthesize DNA and RNA, the cell requires not only pyrimidine nucleo-
tides but also purine nucleotides, and these are needed in roughly equal proportions. Purine
synthesis occurs through a different pathway, but interestingly the final product, the purine
nucleotide adenosine triphosphate (ATP), is a potent activator of the enzyme ATCase. This is
logical, since when the cell contains high concentrations of purine nucleotides it will require
equally high concentrations of pyrimidine nucleotides in order for these two types of nucleotide
to combine to form the polymers DNA and RNA. Thus ATCase is able to regulate the produc-
tion of pyrimidine nucleotides within the cell according to cellular demand, and also to ensure
that pyrimidine nucleotide synthesis is synchronized with purine nucleotide synthesis—an ele-
gant biochemical mechanism for the regulation of an extremely important metabolic process.
There are some rare, although important, cases of monomeric enzymes that have only one
substrate-binding site but are capable of demonstrating the sigmoidal reaction kinetics charac-
teristic of allosteric enzymes. Particularly noteworthy in this context is the monomeric enzyme

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P.K. Robinson 21

glucokinase (also called hexokinase IV), which catalyses the phosphorylation of glucose to glu-
cose-6-phosphate (which may then either be metabolized by the glycolytic pathway or be used
in glycogen synthesis). It has been postulated that this kinetic behaviour is a result of individual
glucokinase molecules existing in one of two forms—a low-affinity form and a high-affinity
form. The low-affinity form of the enzyme reacts with its substrate (glucose), is then turned into
the high-affinity form, and remains in that state for a short time before slowly returning to its
original low-affinity form (demonstrating a so-called slow transition). Therefore at high sub-
strate concentrations the enzyme is likely to react with a second substrate molecule soon after
the first one (i.e. while still in its high-affinity form), whereas at lower substrate concentrations
the enzyme may transition back to its low-affinity form before it reacts with subsequent sub-
strate molecules. This results in its characteristic sigmoidal reaction kinetics.

Origin, purification and uses of enzymes

Enzymes are ubiquitous
Enzymes are essential components of animals, plants and microorganisms, due to the fact that
they catalyse and co-ordinate the complex reactions of cellular metabolism.
Up until the 1970s, most of the commercial application of enzymes involved animal and
plant sources. At that time, bulk enzymes were generally only used within the food-processing
industry, and enzymes from animals and plants were preferred, as they were considered to be
free from the problems of toxicity and contamination that were associated with enzymes of
microbial origin. However, as demand grew and as fermentation technology developed, the
competitive cost of microbial enzymes was recognized and they became more widely used.
Compared with enzymes from plant and animal sources, microbial enzymes have eco-
nomic, technical and ethical advantages, which will now be outlined.

Economic advantages
The sheer quantity of enzyme that can be produced within a short time, and in a small produc-
tion facility, greatly favours the use of microorganisms. For example, during the production of
rennin (a milk-coagulating enzyme used in cheese manufacture) the traditional approach is to
use the enzyme extracted from the stomach of a calf (a young cow still feeding on its mother’s
milk). The average quantity of rennet extracted from a calf ’s stomach is 10 kg, and it takes sev-
eral months of intensive farming to produce a calf. In comparison, a 1 000-litre fermenter of
recombinant Bacillus subtilis can produce 20 kg of enzyme within 12 h. Thus the microbial
product is clearly preferable economically, and is free from the ethical issues that surround the
use of animals. Indeed, most of the cheese now sold in supermarkets is made from milk coagu-
lated with microbial enzymes (so is suitable for vegetarians).
A further advantage of using microbial enzymes is their ease of extraction. Many of the
microbial enzymes used in biotechnological processes are secreted extracellularly, which
greatly simplifies their extraction and purification. Microbial intracellular enzymes are also
often easier to obtain than the equivalent animal or plant enzymes, as they generally require
fewer extraction and purification steps.
Animal and plant sources usually need to be transported to the extraction facility,
whereas when microorganisms are used the same facility can generally be employed for pro-
duction and extraction. In addition, commercially important animal and plant enzymes are

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22 Essays in Biochemistry volume 59 2015

often located within only one organ or tissue, so the remaining material is essentially a waste
product, disposal of which is required.
Finally, enzymes from plant and animal sources show wide variation in yield, and may
only be available at certain times of year, whereas none of these problems are associated with
microbial enzymes.

Technical advantages
Microbial enzymes often have properties that make them more suitable for commercial exploi-
tation. In comparison with enzymes from animal and plant sources, the stability of microbial
enzymes is usually high. For example, the high temperature stability of enzymes from thermo-
philic microorganisms is often useful when the process must operate at high temperatures (e.g.
during starch processing).
Microorganisms are also very amenable to genetic modification to produce novel or
altered enzymes, using relatively simple methods such as plasmid insertion. The genetic
manipulation of animals and plants is technically much more difficult, is more expensive and
is still the subject of significant ethical concern, especially in the U.K.

Enzymes may be intracellular or extracellular

Although many enzymes are retained within the cell, and may be located in specific subcellular
compartments, others are released into the surrounding environment. The majority of enzymes in
industrial use are extracellular proteins from either fungal sources (e.g. Aspergillus species) or bac-
terial sources (e.g. Bacillus species). Examples of these include α-amylase, cellulase, dextranase,
proteases and amyloglucosidase. Many other enzymes for non-industrial use are intracellular and
are produced in much smaller amounts by the cell. Examples of these include asparaginase, cata-
lase, cholesterol oxidase, glucose oxidase and glucose-6-phosphate dehydrogenase.

Enzyme purification
Within the cell, enzymes are generally found along with other proteins, nucleic acids, polysac-
charides and lipids. The activity of the enzyme in relation to the total protein present (i.e. the
specific activity) can be determined and used as a measure of enzyme purity. A variety of
methods can be used to remove contaminating material in order to purify the enzyme and
increase its specific activity. Enzymes that are used as diagnostic reagents and in clinical thera-
peutics are normally prepared to a high degree of purity, because great emphasis is placed on
the specificity of the reaction that is being catalysed. Clearly the higher the level of purifica-
tion, the greater the cost of enzyme production. In the case of many bulk industrial enzymes
the degree of purification is less important, and such enzymes may often be sold as very crude
preparations of culture broth containing the growth medium, organisms (whole or frag-
mented) and enzymes of interest. However, even when the cheapest bulk enzymes are utilized
(e.g. proteases for use in washing powders), the enzyme cost can contribute around 5–10% of
the final product value.

Pretreatment
At the end of a fermentation in which a microorganism rich in the required enzyme has been
cultured, the broth may be cooled rapidly to 5°C to prevent further microbial growth and sta-
bilize the enzyme product. The pH may also be adjusted to optimize enzyme stability. If the

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P.K. Robinson 23

enzyme-producing organism is a fungus, this may be removed by centrifugation at low speed.

If the enzyme source is bacterial, the bacteria are often flocculated with aluminum sulfate or
calcium chloride, which negate the charge on the bacterial membranes, causing them to clump
and thus come out of suspension.

Treatment
Extracellular enzymes are found in the liquid component of the pretreatment process.
However, intracellular enzymes require more extensive treatment. The biomass may be con-
centrated by centrifugation and washed to remove medium components. The cellular compo-
nent must then be ruptured to release the enzyme content. This can be done using one or more
of the following processes:

• ball milling (using glass beads)

• enzymic removal of the cell wall
• freeze–thaw cycles
• liquid shearing through a small orifice at high pressure (e.g. within a French press)
• osmotic shock
• sonication.

Separation of enzymes from the resulting solution may then involve a variety of separa-
tion processes, which are often employed in a sequential fashion.
The first step in an enzyme purification procedure commonly involves separation of the
proteins from the non-protein components by a process of salting out. Proteins remain in
aqueous solution because of interactions between the hydrophilic (water-loving) amino acids
and the surrounding water molecules (the solvent). If the ionic strength of the solvent is
increased by adding an agent such as ammonium sulfate, some of the water molecules will
interact with the salt ions, thereby decreasing the number of water molecules available to inter-
act with the protein. Under such conditions, when protein molecules cannot interact with the
solvent, they interact with each other, coagulating and coming out of solution in the form of a
precipitate. This precipitate (containing the enzyme of interest and other proteins) can then be
filtered or centrifuged, and separated from the supernatant.
Since different proteins vary in the extent to which they interact with water, it is possi-
ble to perform this process using a series of additions of ammonium sulfate, increasing the
ionic strength in a stepwise fashion and removing the precipitate at each stage. Thus such
fractional precipitation is not only capable of separating protein from non-protein compo-
nents, but can also enable separation of the enzyme of interest from some of the other pro-
tein components.
Subsequently a wide variety of techniques may be used for further purification, and steps
involving chromatography are standard practice.
Ion-exchange chromatography is often effective during the early stages of the purifica-
tion process. The protein solution is added to a column containing an insoluble polymer (e.g.
cellulose) that has been modified so that its ionic characteristics will determine the type of
mobile ion (i.e. cation or anion) it attracts. Proteins whose net charge is opposite to that of the
ion-exchange material will bind to it, whereas all other proteins will pass through the column.
A subsequent change in pH or the introduction of a salt solution will alter the electrostatic
forces, allowing the retained protein to be released into solution again.

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Gel filtration can be utilized in the later stages of a purification protocol to separate mol-
ecules on the basis of molecular size. Columns containing a bed of cross-linked gel particles
such as Sephadex are used. These gel particles exclude large protein molecules while allowing
the entry of smaller molecules. Separation occurs because the larger protein molecules follow a
path down the column between the Sephadex particles (occupying a smaller fraction of the
column volume). Larger molecules therefore have a shorter elution time and are recovered first
from the gel filtration column.
Affinity chromatography procedures can often enable purification protocols to be
substantially simplified. Typically, with respect to enzyme purification, a column would
be packed with a particulate stationary phase to which a ligand molecule such as a sub-
strate analogue, inhibitor or cofactor of the enzyme of interest would be firmly bound. As
the sample mixture is passed through the column, the enzyme interacts with, and binds,
to the immobilised ligand, being retained within the column as all of the other compo-
nents of the mixture pass through the column unrewarded. Subsequently a solution of the
ligand is introduced to the column to release (elute) and thereby recover the bound
enzyme from the column in a highly purified form.
Nowadays numerous alternative affinity chromatography procedures exist that are able to
separate enzymes by binding to areas of the molecule away form their active site. Advances in
molecular biology enable us to purify recombinant proteins, including enzymes, through affin-
ity tagging. In a typical approach the gene for the enzyme of interest would be modified to
code for a further short amino acid sequence at either the N- or C- terminal. For example, a
range of polyhistidine tagging procedures are available to yield protein products with six or
more consecutive histidine residues at their N- or C- terminal end. When a mixture contain-
ing the tagged protein of interest is subsequently passed through a column containing a nickel-
nitrilotriacetic acid (Ni-NTA) agarose resin, the histidine residues on the recombinant protein
bind to the nickel ions attached to the support resin, retaining the protein, whilst other protein
and non-protein components pass through the column. Elution of the bound protein can then
be accomplished by adding imidazole to the column, or by reducing the pH to 5-6 to displace
the His-tagged protein from the nickel ions.
Such techniques are therefore capable of rapidly and highly effectively isolating an
enzyme from a complex mixture in only one step, and typically provide protein purities of up
to 95%. If more highly purified enzyme products are required, other supplemental options are
also available, including various forms of preparative electrophoresis e.g. disc-gel electrophore-
sis and isoelectric focusing.

Finishing of enzymes
Enzymes are antigenic, and since problems occurred in the late 1960s when manufacturing
workers exhibited severe allergic responses after breathing enzyme dusts, procedures have now
been implemented to reduce dust formation. These involve supplying enzymes as liquids wher-
ever possible, or increasing the particle size of dry powders from 10 μm to 200–500 μm by
either prilling (mixing the enzyme with polyethylene glycol and preparing small spheres by
atomization) or marumerizing (mixing the enzyme with a binder and water, extruding long fil-
aments, converting them into spheres in a marumerizer, drying them and covering them with
a waxy coat).

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