2.1.

1 - Microscopes - magnification and resolution

LM - - EM
EM
-
2
LM
LM
EM LM

The cell is the basic unit of an organism and consists of a jelly-like material surrounded
by a cell membrane. It can be seen with a light microscope (LM) but many of the
structures within a cell - organelles - can only be seen clearly with an electron microscope
(EM). That is partly because an EM has a greater magnifying power (ability to enlarge
something). However, increasing only magnification has its limits because at some point
magnification reveals nothing more - the details only look bigger and vaguer. This is
because if 2 objects are a distance of less than half the wavelength of light apart, they
cannot be distinguished as separate by a LM. Any object less than half the wavelength of
light in size will not be seen at all by a LM. Using electrons instead of light means that
the illumination has a much shorter wavelength than light. This is good because minute
detail can be detected. We say that an EM has a bigger resolving power (bigger
resolution) than an LM.

2.1.2 (Prokaryotic and eukaryotic cells)

There are 2
basic cell types:
Prokaryotic:
bacteria and
cyanobacteria
(which used to be called blue-green algae). Eukaryotic: all other cells, such as protoctista,
fungi, plant and animal cells.

0.5-5um 40 um 15 um

DNA DNA

70S 80S 80S

2.2.1 Cytosol and Endoplasmic Reticulum, ER

ER

2
ER - RERSERSER

SER
RNA DNA

RER
Cytoplasm refers to the jelly-like material with organelles in it. If the organelles were
removed, the soluble part that would be left is called the cytosol. It consists mainly of
water with dissolved substances such as amino acids in it. Also present in the cytosol are
larger proteins and enzymes used in reactions within the cell. Running through the
cytosol is endoplasmic reticulum (ER), a system of flattened cavities lined by a thin
membrane. It is the site of the synthesis of many substances in the cell and so provides a
compartmentalised area in which this takes place. The cavities also function as a
transporting system whereby substances can move through them from one part of the cell
to another. There are 2 types of ER - rough (RER) and smooth (SER). SER obviously
looks as though it has a smooth surface. It is where lipids and steroids are made so you
would expect there to be a lot of SER in liver cells where lipid is metabolised. RER looks
rough on the surface because it is studded with very small organelles called ribosomes.
Ribosomes are made of RNA and protein and are the site of protein synthesis (see DNA
and Genetic Code). There may be free ribosomes in the cytoplasm as well, which also are
the site of protein synthesis. The proteins (which include enzymes) that are synthesised
then move into the cavities of the RER to be transported.

2.2.2 Golgi apparatus

The Golgi apparatus is a series of flattened layers of plate-like membranes. The proteins
that are made by the RER for export from the cell are pinched off at the end of the cavity
of the RER, so that a layer of membrane surrounds them. The whole structure is called a
vesicle. This vesicle will move through the cytosol and fuse with the membrane of the
Golgi apparatus. In the cavity of the Golgi apparatus, the vessel proteins are modified for
export - for example, by having a carbohydrate added to the protein. At the end of a Golgi
cavity, the secretory product is pinched off so that the vesicle containing the substance
can move through the cytosol to the cell surface membrane. The vesicle will fuse with
this membrane and so release the secretory product. If the vesicle contains digestive
enzymes, it is called a lysosome. Lysosomes may be used inside the cell during
endocytosis, or to break-down old, redundant organelles.

2.2.3 Mitochondria

1000
 2

ATPATP

A typical cell
may contain
1,000
mitochondria,
though some
will contain
many more.
Generally,
they are sausage-shaped organelles whose walls consist of 2 membranes. The inner
membrane is folded inwards to form projections called cristae. Inside this is the matrix.
Most of the reactions for aerobic respiration take place in the mitochondria so it is an
incredibly important organelle. During respiration, ATP is produced, which is used to
provide energy for the cells reactions. Most of the ATP is produced on the inner
mitochondrial membrane.It is highly folded so there is maximum surface area available.

2.2.4 Cell wall and chloroplasts

- -

These are only found in plant cells. Chloroplasts will be discussed in photosynthesis -
but, like the mitochondria - they have an envelope of two membranes making up the
outer "wall".They have pairs of membranes called thylakoids arranged in stacks, each
stack being called a granum. Connecting different grana together are inter-granal
thylakoids. Surrounding the internal membranes, inside the envelope is the stroma. The
reactions of photosynthesis take place in the membranes and stroma of the chloroplast.
The cell wall is rigid and made of cellulose fibres running through a mixture of other
polysaccharides (more complex sugars) such as pectins and hemicelluloses. The sticky
middle lamella that holds next-door cells together is made of calcium pectate and
magnesium pectate. In young cells, the cellulose fibrils of the primary cell wall run
parallel to each other. In older cells, a secondary cell wall may be laid down where the
fibres are all parallel to each other, but at a different angle to those of the primary cell
wall. The cell wall is fully permeable unless a substance called lignin is deposited in the
cellulose layers. Lignin makes the cell wall very strong and resistant to strain but it also
makes it impermeable. If all the gaps between the fibres are filled in, the wall becomes
completely impermeable and the cell will die.

2.2.5 Nucleus

2 -
2 ER
DNA RNA

The nucleus is separated from the surrounding cytoplasm by the double membrane
around it, the nuclear envelope. This regulates the flow of substances into and out of the
nucleus. At some points around the nucleus, the 2 membranes fuse to create nuclear pores
- these are channels through which substances can move. The outer of the 2 membranes is
continuous with the ER. Within the nuclear envelope is the nucleoplasm. In this are
suspended thread-like chromosomes (for chromosome structure see DNA and Genetic
Code). Another structure within the nucleus is the nucleolus. The RNA, which will be
made into ribosomes, is synthesised in the nucleolus.

2.2.6 Other organelles

Vacuole: a fluid-filled space in the cytoplasm surrounded by a membrane called the

tonoplast. It contains a solution of sugars and salts called the cell sap.

Microtubules: hollow rod-like structures with walls of tubulin protein. They provide the
structural support of cells and can aid transport through the cell.

Microfilaments: rod-like structures made of contractile protein. Again, like microtubules,

provide support and aid movement.

Centrioles: a pair of short hollow cylinders, usually found near the nucleus of an animal
cell. They are involved in the formation of spindle fibres used in mitosis (see
Reproduction and Cell Cycle Learn-it).

Cilia: hollow tubes extending outside some cells. They move fluid, which is outside the
cell - for example, ciliated cells lining the respiratory tract move mucus, away from the
lungs.

Flagella: similar to cilia, though longer. Used in the movement of the whole cell. The
only structure like this in humans is the tails of the sperm.

2.2.7 Investigating the function of cell organelles

??

To obtain reliable information about the activity of an organelle, it is necessary to isolate

it and test it individually. First the cells are broken open or cell fractionation occurs to
produce a homogenate or suspension. This is done using a blender with the cells in an
isotonic, cold solution. Because the solution is isotonic, the organelles neither gain, or
loose water by osmosis and as it is cold, the action of enzymes, which might damage the
organelles, is prevented. Differential centrifugation of the suspension is then carried out.
A tube containing the suspension is spun in a centrifuge at a speed, which causes the
heaviest organelles to be thrown to the bottom, forming a sediment. The other lighter
organelles remain floating in the clear supernatant fluid above the sediment. The
sediment may be removed and the activity of the heaviest organelles such as the nucleus,
determined. The supernatant may then be spun at a faster speed so that lighter organelles
like the mitochondria sediment out.

It is important that the cell is supplied with all the substances it needs (e.g. oxygen) and
that waste substances (e.g. carbon dioxide), or substances for export, leave the cell. There
are various processes by which this can happen...

2.4.1 Diffusion

This is the process that is used in oxygen entering a cell, and carbon dioxide leaving.
These molecules will move from where they are at a high concentration to where they are
at a lower concentration. i.e. they diffuse down a concentration gradient. The blood
system in humans continually brings more oxygen to the cell and takes carbon dioxide
away. This maintains a high concentration gradient. Since the movement is always down
the concentration gradient, it requires no energy. The small molecules pass from one side
of the membrane to the other by moving between the lipid molecules.

2.4.2 Ficks Law

Fick

The larger the area and difference in concentration and the thinner the surface, the
quicker the rate. So, for example, in the lung the surface area is made very large by the
presence of many alveoli. The difference in concentration is maintained by breathing,
which brings in air with a high oxygen concentration and removes the air with a high
carbon dioxide concentration and by a good blood supply. The capillaries surrounding the
alveoli take away the oxygenated blood and replace it with blood with a high carbon
dioxide concentration. The walls of the alveoli are only one cell thick, so the surface
across which diffusion occurs is thin and the rate is high. In plants, a good example
would be root hair cells. They have a very large surface area due to the drawing out of the
cytoplasm to produce a very fine root hair. Water continues to enter the root by osmosis
because there is a high concentration of mineral salts in the cells and the water is moved
up the plant by the xylem. Water only has to penetrate one cell in order to enter the plant
and so again the rate of diffusion is high.

2.4.3 Osmosis

If two solutions are separated by a semi-permeable membrane, which only allows certain
sized molecules through (as in a plasma membrane), there will be a net (overall)
movement of the water molecules, from the less concentrated solution (the one with more
water molecules), to the solution which is more concentrated (has more solute
molecules). This is because as in ordinary diffusion the molecules move to even-out any
difference in concentration. However, because of the semi-permeable membrane, which
does not allow the larger solute molecules to cross, only the water molecules can move.
The water molecules will continue to cross the semi-permeable membrane until an
equilibrium is reached, where the two solutions are of equal concentration.
2.4.4 Water potential

PSI

This is a measure of the tendency of water molecules to move from one place to another.
The symbol used for water potential is the Greek letter psi, . Water always moves from
a region of higher water potential to one of lower water potential, or down the
concentration gradient. So we can define osmosis as the movement of water molecules
from a region of higher water potential to a region of lower water potential through a
semi-permeable membrane.

2.4.5 Solute potential and pressure potential

Solute potential and pressure potentialThe water potential of a cell is dependent upon the
combination of its solute and pressure potentials. The water potential of pure water is
zero and since adding solutes lowers water potential, they make the water potential less
than zero, i.e. negative. The more solute, the more negative the water potential becomes.
The amount that the solute molecules lower the water potential is called the solute
potential. It always has a negative value and is given the symbol, s. Pressure also has a
role to play in determining water potential. The greater the pressure inside a cell, the
greater the tendency will be for water to leave it. This contribution to water potential is
called the pressure potential. It always has a positive value because it increase water
potential and is given the symbol p.

2.4.6 Osmosis in animal and plant cells

In animal cells:

Water potential = Solute potential

Or:

= s

So in plant cells the equation used to calculate the water potential of a cell is therefore:

Water potential = Solute potential + Pressure potential

Or:

= s + p

f the water potential surrounding an animal cell is higher than that of the cell, it will gain
water, swell and burst. If the surrounding solutions water potential is lower than that of
the cell, it will loose water and shrivel up. This is why it is so important to maintain a
constant water potential inside the bodies of animals. Pressure potential is important in
plant cells because they are surrounded by a cell wall which, is strong and rigid. When
water enters a plant cell, its volume increases and the living part of the cell presses on the
cell wall. The cell wall gives very little and so pressure starts to build up inside the cell.
This has the tendency to stop more water entering the cell and also stops the cell from
bursting. When a plant cell is fully inflated with water, it is called turgid. If a plant cell is
placed in a solution with a lower water potential, it will loose water. The living part of the
cell or protoplast will shrink and pull away from the cell wall. At this point the pressure
potential is zero and so the water potential of the cell is equal to its solute potential. This
process is called plasmolysis and the cell is said to be plasmolysed. The point at which
the protoplast is just about to pull away from the cell wall is called incipient plasmolysis.
2.4.7 Facilitated diffusion

If charged particles or large molecules are to move across the membrane, another process
needs to be found, as they are less soluble (or even insoluble) in lipid. They move
through protein-lined pores.

Channel proteins

These line a water-filled pore in the membrane so water-soluble molecules can easily pass
through. Different channels allow different substances to pass through (the channels are
selective). Some channels are gated (they will only open when appropriately stimulated).
Carrier proteins: In this case, the substance actually combines with a protein and is
carried from one side of the membrane to the other. (The exact details of this process
remain unclear.) These proteins are specific for a particular substance. In both these cases,
substances are moving down the concentration gradient so no energy is required.

2.4.8 Active transport

Sometimes substances need to be moved from where they are at a lower concentration to
where they are at a higher concentration - against the concentration gradient. This allows
cells to take up essential molecules even when they are at a low concentration outside.
Because molecules are moved against the concentration gradient, it requires energy. It is
thought that active transport uses carrier proteins similar to those involved in facilitated
diffusion.
2.4.9 Endocytosis and exocytosis

If very large molecules or groups of molecules need to enter or exit a cell, they do so
using vesicles. The material to be transported out of the cell is surrounded by membrane.
The vesicle will fuse with the cell surface membrane and the contents leave. This is called
exocytosis (see Golgi apparatus earlier). Materials entering the cell can do so when the
plasma membrane invaginates to surround the material. The membrane seals off to form a
vesicle, which can then move into the cell. This is endocytosis. If the material is fluid,
minute vesicles are formed. This type of endocytosis is called pinocytosis. If the material
is relatively large, and is digested by enzymes after fusion of the vesicle with a lysosome,
it is called phagocytosis. This occurs in white blood cells that ingest bacteria and other
foreign bodies.

1.Carbohydrates

Carbohydrates contain 3 elements

1. Carbon, C

2. Hydrogen, H

3. Oxygen, O

Carbohydrates are found in one of three forms

1.1 Monosaccharides

General formula

3 9 (CH2O)
(CH2O)n where n is a number between 3 and 9. They are classified
according to the number of carbon atoms. The monosaccharides you will have to know
fall into these categories:
C=3=

C=4=

C=5= ATP

C=6=

C C All but one

carbon atom have an alcohol (OH) group attached. The remaining carbon atom has an
aldehyde or ketone group attached

1 5 1 6

1 OH

Due to the bond angles between the carbon atoms, it is possible for pentoses and hexoses
to form stable ring structures. The carbon atoms are numbered 1 to 5 in pentoses and 1 to
6 in hexoses. Depending on the orientation of the OH group on carbon 1, the
monosaccharide can have either or configurations.

1.2 Disaccharides and glycosidic bonds

1
4 OH

H2O 1,4 -
-1,4- - 1,4 -

These are formed when two monosaccharides are condensed together. One
monosaccharide loses an H atom from carbon atom number 1 and the other loses an OH
group from carbon 4 to form the bond. The reaction, which is called a condensation
reaction, involves the loss of water (H2O) and the formation of an 1,4-glycosidic bond.
Depending on the monosaccharides used, this can be an -1,4-glycosidic bond or a -1,4-
glycosidic bond.
 H OH

Sucrose is used in many plants for transporting food reserves, often from the leaves to
other parts of the plant. Lactose is the sugar found in the milk of mammals and maltose is
the first product of starch digestion and is further broken down to glucose before
absorption in the human gut.

PH

All monosaccharides and some disaccharides including maltose and lactose are reducing
sugars. These can be tested for, by adding Benedicts reagent to the sugar and heating in a
water bath. If a reducing sugar is present, the solution turns green, then yellow and finally
produces a brick red precipitate. Non-reducing sugars can also be tested for using
Benedicts reagent but first require addition of an acid and heating to hydrolyse (break
apart) the sugar. The acid must then be neutralised using an alkali like sodium hydroxide
before carrying out the test as described above.

1.3 Polysaccharides

- Helix
1,4 - 6
300

-
1,4 - - 1,6 -

- 1 ,4 -

1.4 Functions of carbohydrates

1.

2.

3.

4.

5.

6.

Substrate for respiration (glucose is essential for cardiac tissues).

Intermediate in respiration (e.g. glyceraldehydes).

Energy stores (e.g. starch, glycogen).

Structural (e.g. cellulose, chitin in arthropod exoskeletons and fungal walls).

Transport (e.g. sucrose is transported in the phloem of a plant).

Recognition of molecules outside a cell (e.g. attached to proteins or lipids on cell surface
membrane).

1.5 Biochemical test

GCSE

Iodine solution or potassium iodide solution can be used to test for the presence of starch.
A positive result changes the solution from an orange-brown to a blue-black colour. -
refer to gcse and title biochemical test for carboydrate.

2.Lipids

Lipids are made up of the elements carbon, hydrogen and oxygen but in different
proportions to carbohydrates. The most common type of lipid is the triglyceride. Lipids
can exist as fats, oils and waxes. Fats and oils are very similar in structure (triglycerides).
At room temperature, fats are solids and oils are liquids. Fats are of animal origin, while
oils tend to be found in plants. Waxes have a different structure (esters of fatty acids with
long chain alcohols) and can be found in both animals and plants.

Lipids are made up of the elements carbon, hydrogen and oxygen but in different
proportions to carbohydrates. The most common type of lipid is the triglyceride. Lipids
can exist as fats, oils and waxes. Fats and oils are very similar in structure (triglycerides).
At room temperature, fats are solids and oils are liquids. Fats are of animal origin, while
oils tend to be found in plants. Waxes have a different structure (esters of fatty acids with
long chain alcohols) and can be found in both animals and plants.

2.1 Triglycerides

3
COOH 14 22 16-18
3 OH 3 3
OH H
3

These are made up of 3 fatty acid chains attached to a glycerol molecule. Fatty acids are
chains of carbon atoms, the terminal one having an OOH group attached making a
carboxylic group (COOH). The length of the chain is usually between 14 and 22 carbons
long (most commonly 16-18). Three of these chains become attached to a glycerol
molecule which has 3 OH groups attached to its 3 carbons. This is called a condensation
reaction because 3 water molecules are formed from 3 OH groups from the fatty acids
chains and 3 H atoms from the glycerol. The bond between the fatty acid chain and the
glycerol is called anester linkage. The 3 fatty acids may be identical or they may have
different structures. In the fatty acid chains the carbon atoms may have single bonds
between them making the lipid saturated. These are usually solid at room temperature and
are called fats. If one or more bonds between the carbon atoms are double bonds, the lipid
is unsaturated. These are usually liquid at room temperature and are called oils.

2.2 Functions of lipids

1. -
2 - O

3.-
4. -
5. -
6. -
7. -

8. -

1. Storage - lipids are non-polar and so are insoluble in water.

1 High-energy store - they have a high proportion of H atoms relative to O atoms

and so yield more energy than the same mass of carbohydrate.
2 Production of metabolic water - some water is produced as a final result of
respiration.
3 Thermal insulation - fat conducts heat very slowly so having a layer under the
skin keeps metabolic heat in.
4 Electrical insulation - the myelin sheath around axons prevents ion leakage.
5 Waterproofing - waxy cuticles are useful, for example, to prevent excess
evaporation from the surface of a leaf.
6 Hormone production - steroid hormones. Oestrogen requires lipids for its
formation, as do other substances such as plant growth hormones.
7 Buoyancy - as lipids float on water, they can have a role in maintaining buoyancy
in organisms.

2.3 Phospholipids

A phosphate-base group replaces one fatty acid chain. It makes this part of the molecule
(the head) soluble in water whilst the fatty acid chains remain insoluble in water. Due to
this arrangement, phospholipids form bilayers (the main component of cell and organelle
membranes).1.4 Enzymes

......

The majority of the reactions that occur in living organisms are enzyme-controlled.
Without them, the rate of the reactions would be so slow as to cause serious, if not fatal,
damage. Without enzymes toxins would soon build up and the supply of respiratory
substrate would decrease.

Enzymes are proteins and thus have a specific shape. They are therefore specific in the
reactions that they catalyse - one enzyme will react with molecules of one substrate.

The site of the reaction occurs in an area on the surface of the protein called the active
site. Since the active site for all molecules of one enzyme will be made up of the same
arrangement of amino acids, it has a highly specific shape.

Generally, there is only one active site on each enzyme molecule and only one type of
substrate molecule will fit into it.

Chymotrypsin and trypsin both catalyse the hydrolysis of peptide bonds but due to their
shapes, the active site of chymotrypsin only splits bonds after an aromatic amino acid
(one containing a ring of atoms) whereas trypsin only splits bonds after a basic or straight
chain amino acid.

This specificity leads to the lock and key hypothesis.

However, it has been discovered that competitors for an active site (similar in shape to the
substrate) could fit even though they are larger than the substrate. This means that the
substrate and active site are a little flexible.

This has lead to the induced fit model...

When the enzyme and substrate form a complex, structural changes occur so that the
active site fits precisely around the substrate (the substrate induces the active site to
change shape). The reaction will take place and the product, being a different shape to the
substrate, moves away from the active site. The active site then returns to its original
shape.

1.4.1 Enzyme controlled reactions

Reactions proceed because the products have less energy than the substrates. However,
most substrates require an input of energy to get the reaction going, (the reaction is not
spontaneous). The energy required to initiate the reaction is called the activation energy.
When the substrate(s) react, they need to form a complex called the transition state before
the reaction actually occurs. This transition state has a higher energy level than either the
substrates or the product. Outside the body, high temperatures often supply the energy
required for a reaction. This clearly would be hazardous inside the body though!
Fortunately we have enzymes that provide an alternative way with a different transition
state and lower activation energy. The rate of the reaction without any external means of
providing the activation energy continues at a much faster rate with an appropriate
enzyme than without it. The maximum rate that any reaction can proceed at will depend,
among other things, upon the number of enzyme molecules and therefore the number of
active sits available.
1.4.2
Factors
affecting the rate of
reaction

Temperature: enzymes work best at an optimum temperature. Below this,

an increase in temperature provides more kinetic energy to the molecules involved. The
numbers of collisions between enzyme and substrate will increase so the rate will too.
Above the optimum temperature, and the enzymes are denatured. Bonds holding the
structure together will be broken and the active site loses its shape and will no longer
work.

pH pH pH

pH: as with temperature, enzymes have an optimum pH. If the pH changes much from
the optimum, the chemical nature of the amino acids can change. This may result in a
change in the bonds and so the tertiary structure may break down. The active site will be
disrupted and the enzyme will be denatured.

Enzyme concentration: at low enzyme concentration there is great competition for the
active sites and the rate of reaction is low. As the enzyme concentration increases, there
are more active sites and the reaction can proceed at a faster rate. Eventually, increasing
the enzyme concentration beyond a certain point has no effect because the substrate
concentration becomes the limiting factor.

-

-

Substrate concentration: at a low substrate concentration there are many active sites that
are not occupied. This means that the reaction rate is low. When more substrate molecules
are added, more enzyme-substrate complexes can be formed. As there are more active
sites, and the rate of reaction increases. Eventually, increasing the substrate concentration
yet further will have no effect. The active sites will be saturated so no more enzyme-
substrate complexes can be formed.

1.4.3 Cofactors

2 Most enzymes require

additional help from cofactors, of which there are 2 main types:

1.4.3.1 Coenzymes

NAD

These are organic compounds, often containing a vitamin molecule as part of their
structure. Coenzymes are not permanently bound to the enzyme but may be temporarily
and loosely bound for the duration of the reaction and then move away once it is
completed. For example NAD, which transfers hydrogen away from one molecule in a
dehydrogenase reaction and takes it to another molecule (see the Respiration Learn-it).

1.4.3.2 Metal ions

most speed up the formation of the enzyme-substrate complex by altering the charge in
the active site e.g. amylase requires chloride ions, catalase requires iron.

1.4.4 Inhibitors

Inhibitors slow down the rate of a reaction. Sometimes this is a necessary way of making
sure that the reaction does not proceed too fast, at other times, it is undesirable.
Reversible inhibitors: Competitive reversible inhibitors: these molecules have a similar
structure to the actual substrate and so will bind temporarily with the active site. The rate
of reaction will be closer to the maximum when there is more real substrate, (e.g.
arabinose competes with glucose for the active sites on glucose oxidase enzyme). Non-
competitive reversible inhibitors: these molecules are not necessarily anything like the
substrate in shape. They bind with the enzyme, but not at the active site. This binding
does change the shape of the enzyme though, so the reaction rate decreases. Irreversible
inhibitors: These molecules bind permanently with the enzyme molecule and so
effectively reduce the enzyme concentration, thus limiting the rate of reaction, for
example, cyanide irreversibly inhibits the enzyme cytochrome oxidase found in the
electron transport chain used in respiration. If this cannot be used, death will occur.

5.Industrial Enzymes

5.1 Industrial uses of enzymes

??

pH

Many of the reactions catalysed by enzymes have commercial uses. Previously, these
reactions were made to happen without enzymes by using heat and/or strong acids but
enzymes offer the following advantages: They are specific in their action and are
therefore less likely to produce unwanted by-products. They are biodegradable and so
cause less environmental pollution. They work in mild conditions i.e. low temperatures,
neutral pH and normal atmospheric pressure, and are therefore energy saving. However,
the last advantage can also be seen as a disadvantage as their conditions must be
stringently controlled or the enzyme may become denatured. To be effective in a
production process the enzyme molecule must be brought into maximum contact with the
substrate molecules. The solutions can be mixed in suitable concentrations or
immobilisation of the enzyme may be used. This involves attaching the enzyme to an
inert surface such as plastic beads and then bringing the surface into contact with a
solution of the substrate. Immobilisation has the advantage that the enzyme molecules
can be used over and over again, with the result that a lot of product can be made from a
relatively small amount of enzyme. An example of the use of immobilisation is in the use
of lactase. This enzyme hydrolyses lactose (milk sugar), into glucose and galactose

5.2 Examples of the industrial uses of enzymes

Perhaps the best known use is that of protease in biological washing powders. This
enzyme helps to break down protein stains such as blood at lower washing machine
temperatures. This means they save energy and are gentler on clothes. Some people are
allergic to biological washing powders but this may be overcome by encapsulating the
enzymes in wax from which they are only released during the wash.

18.1.1 DNA (Evidence for the structure of DNA)

DNA

1) DNA

2 DNA

DNA AT GC

3 X DNA
X X
X -

Many observations contributed to the evidence from which the structure of DNA was
eventually deduced by Watson and Crick: 1. Chemical analysis. Analytical techniques of
pure DNA revealed the basic constituent molecules, but did not show how they were
joined together. 2. Chargaffs work on base equivalence. Chargaff analysed the DNA
from a wide variety of organisms and found that the ratio between Purines and
Pyrimidines was constant. He also showed that the ratios of Adenine to Thymine and of
Guanine to Cytosine were consistent.Later this indicated the A-T and G-C bonding in
DNA. 3. Franklin and Wilkins work on X-ray crystallography. DNA is a very ordered
molecule and has a consistent symmetry. The technique of X-ray crystallography can
reveal the pattern of such regular molecules. A beam of X-rays passing through the
molecule is scattered by the atoms to give a distinctive X-ray diffraction pattern, which
may be photographed and measured.

18.1.2 RNA The structure of RNA

18.1.2.1 RNA

RNA DNA

1
2
T U
3 DNA
4 RNA RNA RNA

18.1.2.2 RNA

1 RNA RNA 80

2 RNA RNA 3-5

RNA RNA 15

3DNA X

Ribonucleic acid (RNA) is also a polynucleotide. The chain of nucleotides is formed in

exactly the same way as in DNA, but the molecule has some very important differences:
It is a single stranded molecule. The pyrimidine Thymine never occurs but is always
replaced by Uracil, another pyrimidine. (Think "No cup of T for U!")It is much smaller
than DNA. It comes in three different forms, ribosomal, transfer and messenger.

Ribosomal RNA is 80% of the total RNA in a cell. It is involved with the formation of
ribosomes and is therefore important as the site of protein synthesis in a cell. Messenger
RNA is 3-5% of the total RNA in a cell, depending on the protein synthesis activity at the
time. It forms in the nucleus and is used to communicate the genetic code in the allele to
the ribosome during protein synthesis.

Transfer RNA is a clover leaf shaped molecule and is up to 15% of the total RNA in the
cell. It is involved in carrying the amino acids through the cytoplasm to their correct
places in a growing polypeptide chain.The DNA X-ray diffraction photographs showed
the molecule was a double helix and also revealed the exact pitch of the helix, together
with the layer distances. Each layer is of course one base pair.

18.1.3 DNA The structure of DNA

1953 DNA
DNA X
DNA

18.1.3.1 What is nucleotides

, - , -

18.1.3.2 DNA Made up of DNA

DNA
DNA
DNA

4 DNA
AGT C

The nucleotides are linked together, the sugar of one with the phosphate of the next: DNA
is the molecule from which the gene alleles on the chromosomes are made. As with all
nucleotides, those in DNA have three parts. These are a pentose sugar called deoxyribose,
aphosphate group and a nitrogenous base. The sugar and the phosphate are exactly the
same in every nucleotide, but the base varies. There are four bases in DNA and each
nucleotide contains one of them. The bases are called Adenine, Guanine, Thymine
andCytosine. (A,G,T and C for short). The nucleotides are joined in a specific order. The
order of the nucleotides means that the bases they contain are in a certain order, it is this
order which forms the genetic code.

DNA 3C 5
C

The sugar and

phosphate join
together to
make the
backbone of the
DNA molecule.
The 3 carbon
on one sugar is joined to the 5 carbon on the next by means of a phosphate bridge, like
this.

5 3 3 5DNA

ATGC

Each time the sugar joins to a phosphate, a molecule of water is eliminated in a

condensation reaction. This sugar-phosphate-sugar bond is called a phosphodiester bond.
The process repeats so that a very long chain of nucleotides is made, a polynucleotide.
Note that the bases protrude from the side of the chain. There will be a spare 5 sugar
atom at one end of the chain and a spare 3 atom at the other. The chain thus has a 3 to 5
direction reading up the page. In DNA a second polynucleotide chain forms next to the
first, but this runs in the opposite direction. The chains are therefore described as
antiparallel. The bases now find themselves opposite one another and bond together with
weak hydrogen bonds. When this occurs Adenine always pairs with Thymine (A-T) and
Guanine with Cytosine (G-C). There is a good reason for this complementary pairing.
Adenine and Guanine both have a double ring structure and are classified chemically as
Purine bases.

10 34A

When the base pairs form, a consistent spacing is obtained between the polynucleotide
chains.

The whole double chained molecule is formed into a double helix spiral, caused by the
bond angles between each base pair. Each complete turn of the spiral includes ten base
pairs. This takes up a distance of 34 Angstrom .

18.3.1 introduction

DNA DNA

DNA

1) DNA DNA
2 DNA DNA

3 DNA
DNA DNA

Since DNA forms the genetic code and that it is known that genes may be inherited, it
follows that DNA must be copied exactly before being incorporated into gametes at
meiosis.

It also follows that all new cells in an organism must gain a copy of the genes at mitosis,
because they are able to continue the characteristic biochemical behaviour of that
organism.

What is the mechanism for this exact copying or replication of the DNA?

Three theories existed:

1. The parent DNA molecule breaks into segments and new nucleotides fill in the gaps
precisely (fragmentationtheory).

2. The complete parent DNA molecule acts as a template for the new daughter molecule,
which is assembled from new nucleotides. The parent molecule is unchanged
(conservative hypothesis).

3. The parent DNA molecule separates into its two component strands, each of which acts
as a template for the formation of a new complementary strand. The two daughter
molecules therefore contain half the parent DNA and half new DNA (semi-conservative
hypothesis).

18.3.2 DNA The semi conservation hypothesis

Meselsohn 1958
15N
DNA 15N 14N

DNA DNA
40000G20 DNA DNA
15N DNA 14N DNA 14N 15N DNA
15N DNA DNA
14N 15N DNA DNA
14N DNA
DNA


The semi conservative hypothesis was shown to be the true mechanism by the work of
Meselsohn and Stahl(1958). In their experiment they grew the bacterium E.coli in the
presence of radioactive 15N until a culture was obtained in which all the DNA was
labelled with 15N. A subculture of this labelled bacterium was than transferred for growth
in the presence of normal 14N. The generation time of E.coli is known, so it was possible
to take samples of this growing subculture after exactly one, two, three generations and
so on. Each sample had its DNA extracted and the isolated DNA was then centrifuged in
a caesium chloride solution (high viscosity) at 40,000G for 20 hours, causing the DNA to
sediment out. The heavier the DNA, the further it moved down the centrifuge tubes. 15N
DNA is heavier than 14N DNA. Mixed14N and 15N DNA is intermediate in mass
between the two. The original 15N DNA moved to the lowest position in the tube. After
one generation all the DNA moved to an intermediate position, indicating the presence of
only mixed 14N and15N DNA. This was because the DNA in this generation contained
one strand of the parent molecule and one new14N strand. Had the conservative
hypothesis been true, two DNA masses would have been visible, one heavy and the other
light.

18.3.3 DNA The process of DNA replication

DNA DNA
(The actual process is simple. To begin with one strand in the
DNA duplex is nicked by the enzyme DNA topoisomerase, allowing part of the molecule
to unravel to form a replication fork. )

DNA

Next, the enzyme DNA helicase splits the two

strands by breaking the hydrogen bonds. This exposes the bases.

DNA DNA 3
5 5 3DNA
DNA DNA RNA
RNA DNA DNA
DNA
S

DNA polymerase enzyme then moves along the exposed bases sequences, creating a new
complementary strand as it goes. DNA polymerase reads the exposed code from the 3 to
the 5 end and therefore assembles the new strand from the 5 to the 3. Several
molecules of DNA polymerase act simultaneously, each assembling a separate section of
the new strand of DNA. Each DNA polymerase is preceded by an RNA polymerase
enzyme, which constructs an RNA primer to guide the action of the DNA polymerase.
These new DNA segments are then joined together by the enzyme DNA ligase. The two
new daughter molecules then coil up again to reform the double helix structure. This
process occurs during the S phase of thecell cycle.

18.4.1 Introduction

DNA
DNA
DNA

=
=
DNA
Beadle and
Tatum

demonstrated that irradiated moulds developed mutations which affected their ability to
synthesise amino acids. One mutation affected only one amino acid synthesis (a critical
enzyme was absent) and was also passed to subsequent generations.

Their work led to the principle of one gene = one enzyme.

Later this principle was modified to one gene = one polypeptide.

It has since has become one cistron = one polypeptide.

A cistron is the shortest length of DNA that can code for a whole polypeptide.

18.4.2 Protein synthesis

DNA
RNA DNA
RNA U T
RNA AU GC

Protein synthesis relies on the effective communication of the coded information held in
the genes to the sites of protein manufacture, the ribosomes in the cytoplasm. Since DNA
is part of larger structures (chromosomes), which are unable to move from the nucleus,
intermediate messenger molecules are needed. These are messenger RNA molecules. To
begin with, the DNA duplex unzips to expose the base sequence on the coding strand.
RNA nucleotides then move in and align themselves according to the rules of base
pairing (A-U and G-C) with U replacing T in the RNA molecule.

18.4.2.1 Transcription:

RNA RNA DNA

35 mRNA 5 3 DNA
mRNA DNA

The RNA is assembled using the enzyme RNA polymerase. This process is called
transcription.

The DNA template strand is read from the 3 to the 5 end and the mRNA is made from
the 5 to the 3 end. During transcription only the coding parts of the DNA are copied (the
exons). Non coding parts or introns are ignored. The completed mRNA molecule
detaches from the DNA template and exits the nucleus via the nuclear pores, moving into
the cytoplasm.

18.4.2.2 Translation

mRNA
polyribosome
mRNA mRNA
mRNA P
A
RNAtRNA tRNA

AUG


The mRNA is now ready for translation, which is organised by the ribosomes, which now
attach themselves to the mRNA. If more than one ribosome attaches then a polysome
(polyribosome) is formed, with the appearance of beads on a string. Each ribosome
controls the formation of one polypeptide. The mRNA is read as triplets of bases
calledcodons. The ribosome attaches to the mRNA by its small subunit. Magnesium ions
are involved in the attachment process. The larger subunit of the ribosome can
accommodate two codons of the mRNA. One is held in the P (peptidyl) site and the other
in the A (aminoacyl) site. Each codon triplet then attracts a complementary triplet
oranticodon. Each anticodon forms part of one transfer RNA (tRNA) molecule. Each
tRNA carries one specific amino acid in the cytoplasm. The anticodon and codon bind
together temporarily by means of hydrogen bonds. This causes two amino acids to be
held next to one another long enough for the formation of a peptide bondbetween them.
The first amino acid in any polypeptide is usually methionine. The codon for this is AUG,
which has come to be known as the initiation codon as a result. The formation of the
peptide bond is catalysed by the enzymepeptidyl transferase, which is an integral part of
the large ribosome subunit.

18.4.2.3 Translocation

tRNA
mRNA
P A tRNA
5 3 mRNA

tRNA mRNA
UAAUAG UGA
64

1962 T4
DNA
mRNA
polyuradylic - U UUU

Once the peptide bond has formed, the first tRNA detaches and travels into the cytoplasm
to pick up another amino acid. The ribosome shifts along the mRNA by exactly one
codon, so that the second codon now occupies the P site and the third the A site. This
movement is a process called translocation. A third tRNA now moves to the correct
position and a second peptide bond forms. This process is then repeated until the
polypeptide is complete. The ribosome moves along the mRNA from the 5 towards the
3 end. The completion of this process of translation is signalled by nonsense or stop
codons. These do not correspond with any tRNA, but signal the ribosome to detach from
the mRNA. The polypeptide is then ready for modification into a specific protein. A stop
codon may be UAA, UAG or UGA. Watson and Crick originally proposed the triplet
theory on the grounds that this is the minimum number which could give at least one
unique combination for each amino acid. There are in fact 64 possible codons, so each
amino acid has more than one to code for it. The genetic code is thus described as being
degenerate. Evidence for the genetic code being in the form of triplets included the work
of Crick (1962). He showed that if single bases were removed from the DNA of T4
bacteriophages, then frame shifts were caused in the translation to polypeptides. The code
was broken by the work of Nirenberg. He synthesised mRNA with known base sequences
and observed the resulting amino acid sequences. For example, mRNA made only of
uracil (polyuradylic acid or poly-U) gives polypeptides made only of phenylalanine. Thus
the codon UUU corresponds with the amino acid phenylalanine. The genetic code is also
universal (the same in all organisms) and non overlapping (triplets are adjacent).