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Higher Biology
Metabolism & Survival
Notes
METABOLIC PATHWAYS AND THEIR CONTROL
Metabolism is the sum of all chemical reactions taking place in a cell. Metabolic pathways are regulated by enzymes
and can be affected by venoms, toxins and poisons.
Metabolic reactions can be divided into two categories: ANABOLIC (synthesis); CATABOLIC (breakdown).
Catabolic pathways bring about the breakdown (degradation)of
complex molecules to simpler ones, releasing energy in the form of
ATP and often producing building blocks.
Examples: aerobic respiration; digestion
Anabolic pathways bring about the biosynthesis of complex molecules
from simpler building blocks and require energy from ATP.
Examples: protein synthesis; photosynthesis
Reversible and irreversible steps
Metabolic pathways can have reversible and irreversible steps. They can also have alternative routes.
A pathway often contains both reversible and irreversible steps e.g. glycolysis, the breakdown of glucose to
pyruvate has reversible and irreversible steps as well as alternative routes.
GLYCOLYSIS
Glycolysis is the first stage in the process of respiration, where
glucose is converted to pyruvate via a series of intermediate
molecules, with different enzymes controlling each step. Some steps
are reversible, some are not.
Glucose
Step 1
enzyme A
intermediate 1
Step 1
Glucose diffuses from a high concentration outside the cell to a low
concentration inside the cell and is then converted to intermediate 1.
This benefits the cell by maintaining a low concentration of glucose
outside the cell therefore allowing glucose to diffuse constantly into
the cell.
Step 2
The conversion of Intermediate 1 to Intermediate 2 is reversible.
If more intermediate 2 is formed than the cell needs for the next step
then some can changed back into intermediate 1 and used in an
alternative pathway, e.g. to build glycogen in animal cells or starch in
plant cells.
Step 3
The conversion of Intermediate 2 to Intermediate 3 is irreversible.
Intermediate 3 will always be converted to pyruvate (through many
further steps).
Step 2
enzyme B
intermediate 2
Step 3
enzyme C
intermediate 3
many enzyme-controlled steps
pyruvate
Alternative routes
Pathways can be modified and contain alternative routes, so steps can be bypassed e.g. in glycolysis
(used when cell has plenty of sugar). The alternative route bypasses the steps above and the glucose is converted to
sorbitol that is eventually converted to pyruvate.
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Higher Biology
Metabolism & Survival
Notes
EFFECTS OF POISONS, TOXINS AND VENOMS ON METABOLIC PATHWAYS
Poisons
Definition Chemicals that can impair
and damage the body; can be
fatal.
Action
Disrupt metabolic pathways.
Examples
Paraquat (respiratory
distress)
Cyanide (inhibits respiratory
enzymes; used in gas
chambers)
Potassium chloride
(interferes with muscle
contraction; USA executions)
Arsenic (interferes with a
respiratory enzyme)
Toxins
Poisons produced by living
organisms
Interfere with metabolic
pathways.
Tetanus toxin (acts on nervous
system and muscle – causes
spasms)
Curare (acts on motor nerves –
paralysis)
Salmonella toxin (inflammation
of gut lining)
Botulinum toxin (neurotoxin –
paralysis)
Venoms
Poisons produced by e.g.
snakes and scorpions that are
transmitted by a bite or sting.
Affect transmission of nerve
impulses causing spasms or
paralysis.
All scorpions and around 25%
of snake species are venomous,
as are some spiders.
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Higher Biology
Metabolism & Survival
Notes
COMPARTMENTALISATION
Cells have different areas or compartments (organelles) for different functions, increasing efficiency.
The cell itself, and all its organelles, are bounded by membranes. The cell membrane separates the internal
contents of the cell from its surroundings and regulates entry and exit of materials. The membranes surrounding
organelles such as mitochondria, chloroplasts and lysosomes keep chemical metabolites together or apart as
required.
Surface area to volume ratio
Small organelles (and folds in the membrane) increase the surface area to volume ratio, leading to a greater surface
area for reactions and a high concentration of reactants, increasing the chance of reaction.
Mitochondrion
The inner membrane is folded into cristae,
increasing the surface area.
The matrix contains enzymes that control
the citric acid cycle (a metabolic pathway
in respiration).
Chloroplast
Lysosomes
The enzymes needed for ATP generation
are bound together on flattened sacs
containing chlorophyll.
The Calvin cycle (a photosynthetic
metabolic pathway) occurs in the fluid
outside the sacs, where the required
enzymes are also present.
Lysosomes are membrane-bound organelles that
contain powerful digestive enzymes.
Their function is to break down large molecules e.g.
proteins, carbohydrates and nucleic acids, for recycling
of their building blocks.
The enzymes must be kept apart from other parts of
the cell so that they do not destroy them. Also, the
enzymes work at around pH5, rather than at the
cytoplasmic pH of 7.2.
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Higher Biology
Metabolism & Survival
Notes
Structure of the plasma membrane
The cell membrane is made of protein and phospholipid
molecules arranged in a FLUID MOSAIC structure.
The phospholipids are arranged in a BILAYER, with their
hydrophilic heads facing outwards, and their
hydrophobic tails facing each other in the middle of the
bilayer.
The constantly moving phospholipid bilayer contains a
mosaic of different protein molecules in, on or through
it.
TRANSPORT ACROSS MEMBRANES
Transport across the membrane can be PASSIVE or ACTIVE.
Passive transport is movement down a concentration gradient (from high to low) and does not require energy.
Active transport requires energy as molecules move against a concentration gradient (from low to high).
PASSIVE TRANSPORT
• DIFFUSION
Particles of a liquid or gas move down a concentration gradient from a region of high concentration to a region of
low concentration until the concentration is equal. Small molecules such as water, oxygen and carbon dioxide can
pass directly through the lipids in the cell membrane. Larger molecules such as glucose enter and leave via the
pores of specific transport proteins (channel-forming proteins).
• OSMOSIS
Cells can gain or lose water by OSMOSIS. Osmosis is the diffusion of water from a region of high concentration to a
region of low concentration across a selectively permeable membrane (a membrane that allows only small
molecules through).
ACTIVE TRANSPORT
Active transport occurs when molecules are moved across the cell membrane against a concentration gradient.
It is carried out by specific transport proteins.
The transport proteins require an energy supply that is provided by the breakdown of ATP (Adenosine
triphosphate) inside the cell.
An example of active transport is the
action of a transport protein called the
sodium-potassium pump.
The same protein pumps sodium ions out
of the cells and potassium ions into the
cells against their concentration gradients.
This causes an electrical gradient that is
needed for muscle contraction and nerve
impulse transmission.
ENZYMES IN THE MEMBRANE
Some of the proteins in the plasma membrane are ENZYMES that catalyse some metabolic processes.
For example, ATP synthase, present in the membrane of mitochondria, chloroplasts and prokaryotes, catalyses the
synthesis of ATP.
Multi-enzyme complexes ensure that steps in a metabolic pathway occur in the correct order.
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Many membrane-bound enzymes are found in the cells of the small intestine, which are involved in the final stages
of digestion and absorption. These enzymes are located on the ‘outside’ of the intestinal cells and break down
small polysaccharides into single sugars, or polypeptides into amino acids. These small molecules can then be
imported into the cells. By embedding these enzymes in the plasma membrane, the final products of digestion are
produced close to the transport proteins that take them into the cell.
ENZYME ACTION
Revision
Enzymes:
• are proteins
• are biological catalysts (speed up chemical
reactions but remain unchanged)
• have an active site on their surface
• some build up (synthesise) small molecules to
make larger molecules (anabolism)
• some break down larger molecules (degrade)
to make smaller molecules (catabolism)
• bind with the substrate at the active site
• are specific (act on one type of substance)
• give the highest rate of reaction under
optimum conditions.
Enzyme active site and substrate molecules are complementary
and fit to form an enzyme-substrate complex. Reaction takes
place and end products are released.
Enzymes are found in the cytoplasm; on membranes; in membrane-bound organelles such as the nucleus,
lysosomes, mitochondria and chloroplasts.
Activation energy and enzyme action
A chemical reaction may involve the joining together of simple molecules into more complex ones or the splitting of
complex molecules into simpler ones. Either way, energy (activation energy) is required to initially break the bonds
in the reactants to form an unstable compound with molecules in a transition state.
In non-living systems a high temperature is
usually needed in order for reactions to proceed.
Enzymes lower the activation energy needed for
the formation of a transition state. Thus
biochemical reactions are able to proceed
rapidly at relatively low temperatures.
Induced fit model
Substrate molecules have an affinity for the active site and are complementary to it. However, the match between
enzyme and substrate may not be an exact fit.
The active site is flexible: when the substrate enters, the
enzyme molecule and the active site change slightly making
the active site fit very closely round the substrate molecule.
The induced fit ensures that the active site comes into very
close contact with the molecules of substrate and increases
the chance of a reaction taking place.
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Orientation of reactants
When the reaction involves two (or more) substrates, the shape
of the active site helps orientate the reactants in the right
position so that a reaction can take place.
1. Reactants orientated to fit active site and held together in an
induced fit.
2. Chemical bonds in reactants are weakened, activation energy
reduced and reaction occurs.
3. Products now have a low affinity for the active site and are
Released, leaving enzyme free to repeat the process.
FACTORS AFFECTING ENZYME ACTION
Enzyme action is affected by temperature and pH. It is also affected by: supply of the substrate(s); concentration of
end-product; presence/absence of an inhibitor *.
Effect of substrate concentration
Low concentration: too few substrate molecules present to
make use of all the active sites on the enzyme molecules.
Increasing substrate concentration: increase in reaction
rate as more active sites are involved.
Further increase in substrate concentration does not
increase rate of reaction further (graph levels off) since all
the active sites are occupied. The enzyme concentration
has become a limiting factor.
Effect of end-product concentration
End product inhibition is negative
feedback used to regulate the production
of a given molecule.
In the example shown, the end product
combines with enzyme 1 to stop the
reaction so there will not be an excess
production of the end product.
Direction of enzyme action
Enzyme 1
Enzyme 2
Enzyme 3
Metabolite W
Metabolite X
Metabolite Y
Metabolite Z
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Once metabolite W becomes available enzyme 1 is activated and converts W to X. When metabolite X becomes
available enzyme 2 is activated and converts X to Y and so on.
Most enzyme reactions are reversible: the actual direction of the reaction depends on the relative concentrations
of reactants and products. In this way a balance of metabolites is always maintained.
[*Note on inhibitors under ‘Control of metabolic pathways by regulation of enzyme action’.]
CONTROL OF METABOLIC PATHWAYS
The role of genes in the control of cell metabolism


Consider this metabolic pathway:
Gene(s)
Gene(s)

Enzyme 1
Metabolite A
Enzyme 2
Metabolite B

Each step is driven by a specific enzyme.
Each enzyme is coded for by a gene or
genes.
If the correct enzyme is present then the
pathway proceeds.
If one enzyme is absent the pathway will
stop.
Metabolite C
Enzyme action can be regulated by the level of gene expression. Some metabolic reactions e.g. respiration are
always required; others may need to operate only in certain circumstances. To avoid wasting resources, the genes
coding for enzymes are ‘switched on’ or ‘off’ as required.
GENETIC CONTROL OF METABOLIC PATHWAYS
Genetic control involves the switching of genes on and off.
One example of a genetic switch was discovered by
French biologist François Jacob and Jaques Monod, who
studied the breakdown of lactose in the bacterium
Escherichia coli (E. coli).
There are hundreds of different strains of E. coli.
Most are harmless and live in the intestines of
mammals. Some can cause gastrointestinal infections.
Lactose metabolism in E.coli
Lactose is a sugar found in milk. E.coli’s normal environment (the gut) usually has glucose but not lactose.
However, E.coli has a gene that enables it to digest lactose should any be present. This gene is only switched on
when lactose is present.
Background facts
 Glucose is used by E. coli for energy release.
 Lactose sugar – found in milk – is composed of
glucose and galactose.
 Lactose is broken down by the enzyme ßgalactosidase.
 E. coli has a gene coding for ß-galactosidase.
 E. coli produces the enzyme only when
lactose is present.
The action of ß-galactosidase on lactose
ß-galactosidase
Lactose
glucose + galactose
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The gene for the enzyme is only switched on when lactose is present - it is otherwise switched off.
This process of switching on a gene only when the enzyme it codes for is required is called ENZYME INDUCTION.
In the case above, lactose is the INDUCER: its presence allows the expression of the genes coding for the enzyme
that will break it down. These genes form part of a section of DNA called an OPERON.
Operons
An operon consists of one or more structural genes
with an adjacent operator gene that controls
it/them.
The operator gene is regulated by a regulator gene
that codes for a repressor molecule. The repressor
prevents the operator gene from being transcribed.
The Lac operon of E. coli
An example of an operon is the lac (lactose) operon of E. coli.
 Structural gene is transcribed and translated into the enzyme ß- galactosidase which breaks down the
sugar lactose.
 Operator gene switches on the structural gene
 Regulator gene controls the functioning of the operator through the production of a ‘repressor protein’
Lactose absent
Operator
mRNA for repressor
protein transcribed
and translated
Repressor
protein
Operator blocked
Repressor protein
binds to operator
gene
structural genes
switched off
No galactosidase
synthesised
Lactose present
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Operator
mRNA for repressor
protein transcribed
and translated
Repressor
protein
Operator is freed
Structural genes
allowing it to switch on
switched on
structural gene
Lactose binds the
repressor molecule
Lactose
enters cell
mRNA for enzyme
transcribed, and then
translated into the protein
-galactosidase
synthesised
Enzyme digests lactose until its supply
runs out.
Repressor then free to bind with
operator and gene switched off.
Lactose is called the inducer because its presence induces synthesis of the enzyme.
Use of ONPG in investigations of the lac operon
ß- galactosidase will also break down a colourless chemical called ONPG:
ß- galactosidase
ONPG
galactose + yellow compound (ortho-nitrophenol)
This is useful in experiments to indicate the activity of ß- galactosidase as the yellow compound is easily seen and its
presence is an indicator of enzyme activity.
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Notes
THE ARA OPERON
E. coli can also use the sugar ARABINOSE in the absence of glucose. As in the case of lactose, arabinose is the
inducer for the enzymes that break it down.
ara operon
Arabinose absent
group of structural genes coding for enzyme
section of
chromosome
Regulator
gene
Transcription and
translation to
form inactive
regulator
molecule
promoter
Regulator binds
to DNA but
remains inactive
Structural genes remain
switched off
No arabinose-digesting
enzyme synthesised
Regulator
molecule
Arabinose present
group of structural genes coding for enzyme
section of
chromosome
Regulator
gene
Transcription and
translation to
form inactive
regulator
molecule
promoter
arabinose combines
with regulator,
changing its shape
and making it act on
promoter
RNA polymerase begins
transcription
Regulator
molecule
Transcription and
translation of arabinosedigesting enzyme
arabinose
enters cell
Transformation of the ara operon
The DNA of E. coli can be artificially transformed e.g. by having particular genes inserted into a plasmid.
The structural genes of the ara operon can be replaced with a gene coding for green fluorescent protein (GFP) and
another that confers resistance to ampicillin. [This is done using pGLO plasmids.]
gene for ampicillin resistance
section of
chromosome
Regulator
gene
promoter
GFP gene in place
of gene for
enzyme
Bacteria transformed in this way will produce GFP instead of arabinose-digesting enzymes (in the presence of
arabinose) and will be ampicillin-resistant. These features allow the operation of the operon to be studied more
easily.
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CONTROL OF METABOLIC PATHWAYS BY REGULATION OF ENZYME ACTION
Some metabolic pathways must operate continuously – their genes are always ‘on’ and the enzymes they code for
are always present in the cell.
Such pathways are controlled by regulation of the rates of reaction of their enzymes by signal molecules and
inhibitors
Signal molecules
A signal molecule causes a gene to be switched on by combining with the product of the regulator gene so that the
structural gene produces the required enzyme.
Signal molecules can be INTRACELLULAR or EXTRACELLULAR.
• Intracellular signal molecules come from within the cell itself.
• Extracellular signal molecules come from outside the cell e.g. adrenaline is made by the adrenal glands and
acts on liver cells to activate the enzyme that converts glycogen to glucose.
Inhibitors
An inhibitor is a substance that reduces the rate of an enzyme-controlled reaction. Inhibitors can occur naturally or
be man-made e.g. drugs, pesticides.
There are three kinds of inhibition: competitive, non-competitive, and feedback inhibition.
Competitive inhibition
Effect of increasing substrate concentration on
competitive inhibition
Competitive inhibitors are
molecules that have a
similar structure to the
substrate and can therefore
fit the enzyme’s active site.
This reduces the rate of
reaction.
Non-competitive inhibition
A non-competitive inhibitor does not combine with
the enzyme’s active site but to a non-active site,
thus changing the shape of the molecule. This
indirectly alters the shape of the active site so that
the substrate molecule cannot bind with it.
Non-competitive inhibitors reduce the amount of
active enzyme and their effect is permanent.
Increasing substrate concentration does not
increase reaction rate in presence of noncompetitive inhibitors.
Examples: cyanide, heavy metal ions, some
insecticides.
Blockage of some active sites by the inhibitor reduces
reaction rate when the substrate concentration is not
at a high level. However, an increase in substrate
concentration increases the chance that it will bind to
the enzyme and reaction rate returns to normal.
Increasing substrate concentration in the presence
of different inhibitors
Competitive: concentration of inhibitor and
substrate control the degree of inhibition.
Non-competitive: concentration of inhibitor alone
controls the degree of inhibition.
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Active and inactive forms of enzyme molecules
Some enzymes are made up of several
polypeptide units, each with its own active and
non-active (allosteric) sites.
The enzyme may exist as an active or inactive
form, each form having a different shape.
The enzyme changes shape if a regulatory
molecule (activator or inhibitor) binds to a nonactive site.
Non-active
site (one of
four)
Active form
Activator: causes the enzyme to take its active
form, stimulating enzyme activity.
Non-competitive inhibitor: causes a change to an
inactive form of enzyme, inhibiting its activity.
Inactive form
End product inhibition
As the concentration of the end product builds up,
some of it binds to molecules of enzyme A.
This slows down the conversion of substrate to the
intermediate 1 metabolite.
Substrate
As the concentration of the end product drops, fewer
molecules of enzyme A are affected and more of
intermediate 1 is converted to intermediate 2 and so
on.
Intermediate 1
Intermediate 2
This is called negative feedback control and avoids the
wasteful conversion and accumulation of
intermediates.
Intermediate 3
Example: Effect of phosphate on the enzyme phosphatase


Phosphatase releases phosphate from its substrate for use in cell metabolism.
Phosphatase acts on the chemical phenolphthalein phosphate, releasing its phosphate.
phosphatase
phenolphthalein phosphate
phenolphthalein + phosphate


Phenolphthalein is pink in alkaline conditions.
This colour change clearly shows the activity of the enzyme (the more activity, the more pink the result).

Increasing phosphate concentration leads to a decrease in enzyme activity – the phosphate acts as an endproduct inhibitor of phosphatase.
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Metabolism & Survival
Notes
CELL RESPIRATION
Respiration is the process occurring in every living cell by which
 chemical energy is released from food (usually carbohydrates, although fats and proteins can also be used
as respiratory substrates);
 the energy is used to regenerate the high-energy molecule adenosine triphosphate (ATP).
Word equation:
glucose + oxygen
carbon dioxide + water
ATP formed
Respiration is a series of catabolic reactions, catalysed by enzymes, in which 6-carbon glucose is oxidised* to form
carbon dioxide. The energy released due to the oxidation of glucose is used to synthesize ATP from adenosine
diphosphate (ADP) and inorganic phosphate (Pi): ADP + Pi
ATP
[*oxidation - loss of hydrogen]
ATP
ATP is a high-energy molecule made during cellular respiration by the addition of inorganic phosphate to ADP. The
energy for this reaction comes from glucose.
high energy bond
ATP structure
ATP is made up of one adenosine and three
phosphate molecules.
The terminal phosphate is held by a high
energy bond: the energy is released when the
bond is broken.
When ATP is broken down into ADP + Pi it releases
its energy. This energy is released as heat but can
also be used in e.g. chemical reactions, muscular
contractions, active transport, nerve impulses, DNA
replication, protein synthesis.
ADENOSINE
P
P
P
3 PHOSPHATE GROUPS
breakdown
releasing energy
ATP
(high energy state)
ADP + Pi
(low energy state)
build-up
requiring energy
ATP as energy carrier
Phosphorylation
Phosphorylation is an enzyme-controlled process by which a phosphate group is added to a molecule
e.g. when low energy ADP and Pi combine to form high energy ATP.
Similarly, if ATP donates a phosphate to a reactant in a metabolic pathway, the reactant becomes phosphorylated
and gains energy, becoming more reactive.
ATP
(high energy state)
Glucose
(low energy state)
ADP + Pi
(low energy state)
Glucose-6-phosphate
(high energy state)
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Notes
ATP in cells
An active cell needs about 2 million molecules of ATP per second to satisfy its energy requirements. This is made
possible by the rapid turnover of ATP: as fast as ATP is broken down to release its energy it is being regenerated
from ADP and Pi (using the energy from respiration).
Only about 50g of ATP is stored in the body at any one time, but the body may be using it up and regenerating it at
about 400g/hr.
ATP synthase
ATP synthesis
The respiratory pathway produces a flow of high-energy
electrons that the cell uses to pump hydrogen ions (H+)
ATP
synthase
across
the inner mitochondrial membrane against a
concentration gradient.
The H+ ions flow back down a concentration gradient across a
membrane protein ATP synthase, causing part of it to rotate.
ATP synthase then catalyses the synthesis of ATP from ADP
and Pi.
ATP synthase molecules are found in the membranes of
mitochondria and chloroplasts.
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THE BIOCHEMISTRY OF RESPIRATION
Respiration can be aerobic (using oxygen) or anaerobic (without oxygen).
Aerobic respiration occurs in three main stages: Glycolysis, the Citric Acid Cycle and the Electron Transport Chain.
GLYCOLYSIS
Glycolysis (‘glucose-splitting’) consists of a series of chemical reactions, each catalysed by a specific enzyme.
It occurs in the cytoplasm and does not require oxygen. During glycolysis, glucose is split into 2 pyruvate molecules.
Energy investment phase
The phosphorylation of
ATP
phosphorylation
at
step
1
intermediates at the beginning of
ADP
the glycolysis pathway uses 2
other metabolic molecules of ATP.
INTERMEDIATE 1
pathways
GLUCOSE
energy
investment
phase
INTERMEDIATE 2
ATP
ADP
phosphorylation at step 3
INTERMEDIATE 3
_________________________________
2NAD
2NADH
energy
pay-off
phase
ADP
ATP
ADP
ATP
Energy pay-off phase
Later reactions in glycolysis result in
regeneration of 4 ATP molecules,
giving a net gain of 2 ATP.
During this phase, H+ ions are
released from glucose by a
dehydrogenase enzyme.
The H ions are passed to a hydrogen
carrier, the coenzyme NAD, forming
NADH.
NADH carries the hydrogen on to a
later stage of respiration, the
electron transport system.
PYRUVATE
After glycolysis
If oxygen is available, pyruvate passes to the next stage in aerobic respiration, the Citric Acid Cycle (also known as
the Krebs Cycle, after its major discoverer).
This cycle of reactions takes place in the matrix of mitochondria.
site of electron transport
chain
site of citric acid cycle
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CITRIC ACID CYCLE
The reactions of the citric acid cycle take place in the matrix of the mitochondrion.
1. Pyruvate is broken down to carbon dioxide and an acetyl group.
2. The acetyl group combines with coenzyme A to form acetyl coenzyme A.
As this happens H ions are released and become joined to NAD forming NADH.
3. The acetyl group of acetyl coenzyme A combines with oxaloacetate to form citrate and enters the citric acid
cycle.
4. After several enzyme-controlled steps oxaloacetate is regenerated.
5. At 3 steps in the cycle dehydrogenase enzymes remove H ions along with associated high-energy electrons.
These H ions and high-energy electrons are passed to the coenzyme NAD to form NADH.
6. A similar reaction at one other step uses the coenzyme FAD which becomes FADH.
7. In addition, ATP is produced at one step and carbon dioxide is released at another two.
Pyruvate
NAD
NADH
CO2
Acetyl group
Coenzyme A
Acetyl coenzyme A
Citrate
2CO2
Oxaloacetate
Citric acid cycle
3NAD
3NADH
FADH2
FAD
ATP
ADP + Pi
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THE ELECTRON TRANPORT CHAIN
The electron transport chain consists of a group of protein molecules attached to the inner membrane of the cristae
of mitochondria. There are many of these chains in a cell.
4
5
6
1. NADH and FADH, from glycolysis and the citric acid cycle, release high-energy electrons and pass them to
the electron transport chains.
2. The electrons begin in a high-energy state. As they flow along a chain of electron acceptors, they release
energy.
3. This is used to pump hydrogen ions across the membrane from the matrix side to the inter-membrane
space to maintain a higher concentration of hydrogen ions.
4. When the hydrogen ions flow back down the concentration gradient to the matrix they pass through
molecules of ATP synthase.
5. This drives this enzyme to synthesise ATP from ADP and Pi.
6. When the electrons come to the end of the electron transport chain they combine with oxygen – the final
hydrogen acceptor. At the same time, the oxygen joins to a pair of hydrogen ions to form water.
Most of the ATP generated by cellular respiration is produced in mitochondria in this way.
In the absence of oxygen the electron transport chains do not proceed and there is no production of ATP except for
that produced during glycolysis.
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SUBSTRATES FOR RESPIRATION
CARBOHYDRATES
starch
glycogen
sucrose
glucose
maltose
Starch and glycogen are broken
down to glucose as required.
Sugars such as maltose and
sucrose can be converted to
glucose or to intermediates in the
glycolysis pathway.
fructose
pyruvate
FATS
glucose
Fats can be broken down into
glycerol and fatty acids.
Glycerol is converted into one of
the intermediates in glycolysis
Fatty acids are eventually
converted into acetyl coenzyme A
for use in the citric acid cycle.
intermediate
Glycerol
Fat
pyruvate
Fatty
acids
acetyl co-A
Citric
acid
cycle
PROTEINS
glucose
pyruvate
amino acid
urea
protein
acetyl co-A
amino acid
Proteins are broken down into
amino acids.
Excess amino acids are
deaminated, forming urea (waste
product) and respiratory pathway
intermediates.
urea
amino acid
intermediate
urea
Citric
acid
cycle
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METABOLIC RATE
Metabolic rate is the quantity of energy used by the body over a given time. It is measured in kilojoules (or
kilocalories).
Metabolic rate can be measured as:
 oxygen consumption per unit time;
 carbon dioxide production per unit time;
 energy production (as heat) per unit time.
[Glucose + oxygen
carbon dioxide + water; energy released
ATP]
Measuring metabolic rate
Metabolic rate can be measured in different ways e.g. using respirometers and calorimeters.
Coloured
water
A respirometer is a chamber with a continuous
airflow that allows the measurement of
differences in oxygen, carbon dioxide and
temperature in air entering and leaving.
A calorimeter measures the heat generated by an organism
by comparing the temperature of water entering and leaving a
well-insulated container.
Basal metabolic rate
Basal metabolic rate (BMR) is the minimum rate of energy release required to maintain essential body processes
when an animal is at complete rest.
BMR is expressed as kilojoules of heat released per square metre of body surface per hour (kJmˉ²hˉˡ).
BMR is low compared to the metabolic rate when the body is exercising.
Comparative metabolic rates
Generally, the greater the mass of an organism the higher its metabolic rate.
Animal
Sea anemone
Octopus
Eel
Frog
Human
Mouse
Hummingbird
Volume of oxygen
consumed
(mm³ g⁻ˡ body mass h¯ˡ)
13
80
128
150
200
1500
3500
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OXYGEN DELIVERY
Oxygen is required for aerobic respiration. High metabolic rates require efficient delivery of oxygen to cells.
Organisms with high metabolic rates need efficient transport systems for the delivery of oxygen.
In vertebrates, oxygen is delivered in blood, pumped by a heart.
Prior knowledge
Main blood vessels involved in the circulation of blood around the body:
 Arteries – carry blood away from the heart (under high pressure).
 Capillaries – smallest blood vessels; exchange nutrients, gases, and waste products between blood and body tissue.
 Veins – carry blood back to the heart (under low pressure).
The heart
The heart has two types of chamber: atria, where blood enters the heart; ventricles , where blood leaves the heart.
Circulatory systems in vertebrates
All vertebrates have closed circulatory systems where the blood is contained in a continuous circuit of blood vessels and is kept
moving by a muscular pump (the heart).
In closed systems a drop in pressure occurs when blood passes through the capillaries because the narrow tubes offer
resistance to the flow of blood.
Single circulatory system (in fish)
Gas exchange occurs in the gills. As water flows over the gill filaments oxygen diffuses down a concentration gradient to the
blood.
Fish have a single circulatory system: blood passes through
the 2-chambered heart only once for each circuit of the
body.
The blood flows to the gills at high pressure but is delivered
to the systemic capillaries at low pressure.
Fish have a two-chambered heart with an
atrium, a ventricle and a valve in between.
Double circulatory system
[Systemic means ‘of the body’]
Double circulatory system
Other vertebrate groups have a double circulatory system: blood passes through the heart twice for each circuit of the body.
Blood is pumped to both the lungs and the body both at high pressure ensuring vigorous flow, making a double circulation
more efficient than single systems.
Double circulatory system: incomplete
Reptile and amphibian circulatory systems are described as
incomplete because there is only one ventricle and some mixing of
oxygenated blood from the lungs and deoxygenated blood from
the body occurs.
In amphibians, some gas exchange occurs through the moist skin,
so blood returning from the body is partially oxygenated.
In most reptiles, the ventricle is partly divided by a septum.
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Double circulatory system: complete
Birds and mammals (and crocodiles!) have complete
circulatory systems: the heart has two atria and two
ventricles completely separated by a septum.
A complete double circulation is the most efficient: it
ensures that oxygenated and deoxygenated blood is not
mixed.
This allows endothermic (‘warm-blooded’) birds and
mammals to access enough oxygen for respiration,
releasing enough heat to keep their bodies warm.
Lung complexity
Essential features of a gas exchange system:
 large surface area;
 moist surface to allow gases to dissolve;


thin structures to allow rapid diffusion into
the tissues of the organism;
good supply of blood vessels.
Amphibians
Amphibians usually exchange gases though skin and mouth lining, only using their lungs when very active.
Lungs: small, thin-walled sacs with few alveoli, if any.
Reptiles and mammals
Reptiles and mammals depend entirely on lungs for gas
exchange. Their lungs have a system of repeatedly
branching tubes ending in many alveoli.
Alveoli have a thin, moist lining and give a large surface
area (100m² in humans) to provide enough oxygen for
aerobic respiration.
Birds
Flight requires a high metabolic rate and consequently a
high supply of oxygen.
Birds have relatively small lungs and several large air sacs
that act as bellows, moving air in one direction through
the lungs.
Birds’ lungs have no alveoli but have parabronchi (tiny
channels) through which oxygenated air passes in one
direction.
.
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Physiological adaptations for low-oxygen niches
There are two main low oxygen niches: high altitudes and deep-diving marine habitats.
Humans at high altitude
We function best at oxygen concentration around 20%. At lower concentrations (e.g. at altitude) we need more red
blood cells (rbcs) in order to function normally.
This is brought about by:
 an increase in the level of erythropoietin, the hormone that stimulates red blood cell production,
 leading to an increase in rbc count,
 thus improving oxygen transport and allowing normal activity.
Deep-diving mammals
Physiological adaptations of mammals such as whales and seals include:
 decrease in heart rate when submerged (less oxygen used by cardiac muscle);
 partial collapse of lungs, forcing air into upper respiratory system and compressing it, making animal less
buoyant;
 high volume of blood per kg body mass;
 high concentration of haemoglobin;
 myoglobin in muscles.
Concentration of atmospheric oxygen over geological time
The Earth’s atmosphere has changed over geological time, with oxygen increasing from none to 1% (possibly
produced by the first photosynthetic cyanobacteria), then to the present 21% (reason not known).
A high level of atmospheric oxygen is necessary to maintain the metabolism of large air-breathing animals.
Oxygen uptake and fitness
Maximum oxygen uptake: the maximum volume of oxygen that a body can take up during gradually-increasing
intense exercise.
VO2 max:
 has been reached when oxygen consumption remains steady;
 is calculated using breathing rate and concentrations of respiratory gases in inhaled and exhaled air
 is measured in cm3kg-1min-1.
VO2 max. score is an indicator of fitness and improves with training.
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METABOLISM IN CONFORMERS AND REGULATORS
The ability of an organism to maintain its metabolic rate is affected by abiotic factors such as temperature and pH.
Organisms can be categorised by the way in which they are able to control their internal environment:
 CONFORMERS are organisms that are directly dependent on their external environment.
 REGULATORS can control their internal environment and maintain a steady state regardless of their
surroundings.
CONFORMERS
Internal environment is directly dependent upon the external environment; not a problem if environment is stable.
Advantage: low metabolic costs
Disadvantages:
• narrow range of ecological niches;
• less adaptable to change.
• many conformers use behavioural responses to help to maintain an optimum metabolic rate e.g. lizards
bask in sunshine to warm up.
REGULATORS
Regulators control their internal environment by physiological means. Regulation of the internal environment
within tolerable means is called HOMEOSTASIS.
Advantage: increases the range of possible ecological niches.
Disadvantage: energy is required to achieve homeostasis.
PHYSIOLOGICAL HOMEOSTASIS
The cells in the body have to be kept in a stable environment even when there are changes in the body’s rate of
activity. This is necessary so that cells can function efficiently.
Maintaining conditions in the body within tolerable limits is called physiological homeostasis.
Homeostasis is necessary for the control of e.g. water concentration, blood sugar level and body temperature.
In homeostasis, the control of the body’s internal environment is brought about by NEGATIVE FEEDBACK CONTROL.
Negative feedback involves a corrective mechanism that opposes any deviation from the normal optimal level
(norm or set point) e.g. rise in body temperature, fall in blood sugar concentration.
Mechanism of negative feedback control
 Changes from the norm (or set
point) are detected by receptors.
 Messages are sent via nerves or
hormones to effectors which
return conditions to normal by
negative feedback control.
Receptors
Corrective
response by
effector
Increase in
factor
Decrease
in factor
Factor
increased
Factor
decreased
Normal
Condition
THERMOREGULATION
Animals can be classified as ECTOTHERMS or ENDOTHERMS according to their ability to regulate their body
temperatures.
Ectotherms are unable to regulate their body temperature and derive their body heat from the environment e.g.
invertebrates, fish, amphibians and reptiles. Some regulate their body temperature by behavioural means e.g. lizard
basking in sunshine.
Endotherms such as mammals and birds derive most of their body heat from their own metabolism. The
temperature of an endotherm’s internal environment must be regulated to ensure that enzyme-controlled
metabolic processes in the body are kept within tolerable limits i.e. 35-40°C.
Endotherm body temperature is independent of external conditions and is under homeostatic control.
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Homeostatic control of temperature
Body temperature is monitored by the HYPOTHALAMUS, a gland in the brain. It receives information about surface
temperature from the thermoreceptors in skin and its own thermoreceptors monitor blood temperature.
The hypothalamus coordinates the response to these signals with priority being given to blood temperatures
(indicate temp. of body core). The hypothalamus sends nerve impulses directly to effectors, triggering corrective
feedback mechanisms.
Effectors
Effector
Increase in temp.
Decrease in temp.
Sweat glands
Skin arterioles
Hair erector
muscles
sweat glands stimulated; heat energy from the
body is used during evaporation of water from
surface of skin and lowers body temperature.
causes vasodilation, leading to increased heat loss
by radiation
body hairs lowered
Skeletal muscles
no increase in activity to cause shivering
Metabolic rate
decrease in metabolic rate
sweating reduced to a minimum
causes vasoconstriction, leading to decrease
in heat loss
causes erector muscles at the base of hairs to
contract; hairs raised from the surface trap a
layer of insulating air and reduce heat loss.
increase in activity and production of
additional heat by shivering
increase in metabolic rate leading to increased
heat production
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METABOLISM AND ADVERSE CONDITIONS
An organism’s environment can change very quickly e.g. rain, wind, sunlight, or more slowly e.g. seasons.
Some changes can cause stress to the organism, when the conditions are beyond the limits of its metabolic rate.
Organisms must maintain a constant internal environment and are therefore either adapted to survive change or
are able to avoid it.
Organisms can have adaptations to help them survive. These can be:
 Structural e.g. body size, appendages, insulation, colour
 Physiological e.g. dormant states such as hibernation (‘winter sleep’); aestivation (‘summer sleep’);
brumation (less deep hibernation in ectotherms); diapause (suspension of growth in insects)
 Behavioural: collective den, snow roost
Dormancy
Some organisms survive extreme conditions by entering dormancy, when their metabolic rate is reduced to the
minimum level necessary to sustain life.
Dormancy can be predictive or consequential.
Predictive dormancy happens in advance of adverse conditions so the organism is prepared.
Examples
• Hibernation in some animals.
• Deciduous trees drop their leaves in Autumn, triggered by decreasing photoperiod, and winter buds remain
closed until Spring.
Consequential dormancy happens after the conditions have arrived e.g. in areas of unpredictable climate. Allows
longer period of activity but may cause death.
Example: many seeds remain dormant during drought and germinate only when sufficient water is available.
Dormancy in seeds
Dormancy can be result of:
• a physical barrier e.g. thick seed coat – has to be broken down by micro-organisms;
• chemical inhibitors – need heavy rain to remove them or long period of cold to halt production.
Both of these can be shown experimentally by scarification of seeds or subjection to cold conditions.
Dormancy ensures that seeds germinate only when conditions are right for plant growth e.g. in the Spring.
Seed banks
Seed banks have been established as a means of preserving seeds from many plant species, especially those that
• are used as food crops (and wild relatives);
• have medicinal properties;
• are rare/threatened.
Seeds must be kept under the best conditions to retain their viability:
 orthodox seeds are desiccation tolerant and are kept at low temperature and low humidity;
 recalcitrant seeds are desiccation intolerant so the whole plant has to kept alive and growing.
Dormancy in animals
Main types: HIBERNATION and AESTIVATION; also DAILY TORPOR
HIBERNATION is a period of inactivity in mammals to enable them to survive winter conditions. Before hibernation,
extra food consumption allows the build-up of a fat store. Occurs in e.g. hedgehogs, dormice.
Physiological changes:
• drop in metabolic rate;
• decrease in body temperature;
• slower heart rate;
• slower breathing rate.
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AESTIVATION allows animals to survive periods of heat and drought by remaining in a state of torpor (deep and
prolonged sleep) with reduced metabolic rate.
Examples
Mediterranean land snails: climb bushes and seal shells with mucus to conserve moisture
Nile crocodiles: stay in burrows, drawing on fat reserves, until the rains arrive.
DAILY TORPOR
Some animals, such as small birds and mammals, enter dormancy on a 24 hour cycle. This is called daily torpor,
when heart, breathing rate and body temperature all decrease.
Small endotherms need a high metabolic rate to maintain body temperature: torpor allows a decrease in overall
energy consumption.
Examples
Hummingbirds feed by day and are in torpor by night whereas nocturnal bats and shrews feed by night and enter
torpor by day.
Migration
Migration is the regular movement of species members from one place to another.
Migration avoids metabolic adversity by expending energy to relocate to a more suitable environment.
The expenditure of energy to relocate is a disadvantage to the organism in the short term but is beneficial in the
long term.
Organisms that migrate include many birds including swallows and corncrakes; mammals such as whales and fish
such as salmon.
Long-distance migrants usually make an annual round trip between the regions e.g. corncrakes breed in Scotland in
Spring but winters in Africa.
Some invertebrates also migrate long distances e.g. Monarch butterflies breed in North America but overwinter in
Mexico (no butterfly makes a return journey as they are short-lived).
Migration study techniques
It is of interest to find out details of migration of species e.g. time of outward and return migration; overwintering
habitat; place returned to; lifespan of animal etc.
Animals are caught and labelled in various ways in order to identify and track them.
• Ringing, usually of birds’ legs, with unique number and contact details; recapture necessary when birds
return, in order to read rings.
• Tagging for small creatures e.g. invertebrates.
• Colour marking of birds – allows them to be seen easily.
• Transmitters glued to body allows satellite tracking; no need to recapture.
Migration triggers
 Photoperiod (day length)
This is the primary trigger for migration in many birds, causing hormonal changes that lead to behavioural and
physiological changes such as restlessness and fat storage.
 The Sun
The position of the Sun is used as a compass to locate the correct direction for migration.
 Stars
Experiments at night (and in planetaria) show that birds respond to particular patterns of stars to direct their
migration.
 Magnetic field
Some birds can sense changes in the Earth’s magnetic field and use these as a compass, perhaps even ‘seeing’ the
magnetic field.
[Birds with magnets attached become disorientated and lose their way easily.]
Some long-distance migrants may use a combination of Sun and magnetic field compasses.
 Internal clock
Many animals have an internal ‘clock’ that allows them to go in the right direction regardless of other stimuli.
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Migration: influences
Migratory behaviour is thought to be influenced by both innate and learned behaviour.
Innate behaviour is inherited from parents to offspring and is likely to be the biggest influence on successful
migration e.g. direction of travel. It is common to all members of a species.
Learned behaviour is gained by experience e.g. specific stop points on a long journey. It may come from parents or
other members of a social group. It is more flexible and has a lesser role in migration.
Experiments
Displacement studies with European starlings have shown that both innate and learned behaviours are used by
birds: young birds use purely innate behaviour and fly according to genetic instructions but experienced birds also
use knowledge of geographical features.
Studies with blackcap warblers show that young birds will fly in the direction typical of their population, showing
innate behaviour.
Cross-fostering of herring gulls (non-migratory) and black-headed gulls (migratory) show that black-headed gulls
migrate even if brought up by herring gulls (innate behaviour) and herring gulls migrated with their foster parents
(learned behaviour).
Migration: genetic control
Genetic studies of the blackcap warbler have shown a link between a gene and migratory behaviour.
The gene codes for a peptide that influences preparations for migration such as metabolic rate and fat usage. It is
thought that this gene causes 3% of migratory behaviour: many other genes must also be involved.
EXTREMOPHILES
An extremophile is an organism that lives in conditions that are ‘extreme’. They are generally they are found in the
domain Archaea.
They have enzymes that are able to function under these unusual conditions, allowing the organisms to thrive in
environments that would be lethal to almost all other species.
Some of these enzymes are used for scientific purposes e.g. heat-tolerant DNA polymerase used in PCR.
Types of extremophiles
Extremophile Environment
Example
Cryophile
cold: temps. as low as -15°C
sea ice; Arctic and Antarctic ice packs.
Thermophile
very hot: 80°C -100°C
deep sea vents and volcanic lakes
Alkaliphile
pH levels 9 or more
soda lakes
Acidophile
pH levels 3 or less
sulphur springs
Halophile
salt conc. at least 0.2M
salt lakes and salt mines
Xerophile
very dry
desert
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ENVIRONMENTAL CONTROL OF METABOLISM
Micro-organisms - introduction
Microbiology is a specialised area of biology that studies organisms that are too small to be seen without
magnification: these are known as microorganisms or microbes.
Microbiology makes up one of the largest and most complex biological sciences because it deals with microbehuman and microbe-environmental interactions.
These interactions are relevant to disease (in both plants and animals), drug therapy, immunology, genetic
engineering, industry, agriculture and ecology.
Microorganisms include archaea, bacteria and some species of eukaryote e.g. yeasts and filamentous fungi.
Some of these species use a wide variety of often cheap substrates for metabolism and produce a range of
products from their metabolic pathways.
Microorganisms are also used in research and industry because of their adaptability, ease of cultivation, speed of
growth and ease of manipulation of metabolism.
Archaea are single-celled and have no nucleus or organelles. While outwardly appearing similar to bacteria, several
of their metabolic pathways are similar to eukaryotes. Many have been classified as extremophiles and the
properties that allowed them to exploit these niches make them of potential use to industry.
Bacteria are divided into three main groups. Each group is further divided until the species level is reached.
Different types within a species are called strains: that is, groups of different cells derived from a single cell.
Many species of bacteria are of economic importance to humans.
Bacteria are involved in the production of yoghurt, cheese, biofuels and many other products and are used in
genetic engineering.
Fungi are eukaryotic cells and can be sub-divided into single-celled yeasts and multicellular moulds.
Some fungi are beneficial and some are harmful.
Beneficial
• decomposers
• essential to many industrial processes that involve fermentation e.g. bread, beer, wine production
• manufacture of antibiotics
Harmful
• major cause of plant disease.
• cause of many animal diseases
Protozoa and Algae
Protozoa are unicellular eukaryotic cells that contain organelles and, like animal cells, lack cell walls.
Algae are plants, often unicellular, growing in a wide variety of moist habitats.
Growth of micro-organisms (cell culture)
Micro-organisms can be cultured relatively easily in a laboratory. They must be given an appropriate growth
medium and the environmental conditions must be carefully controlled to ensure successful growth.
Growth media
A growth medium is a solid or liquid substance used to grow, transport or store micro-organisms.
A liquid medium is called broth and solid media are made of agar jelly with added nutrients.
Growth media can be composed of specific substances or can contain complex ingredients such as beef extract.
Two types of media are commonly used:
• complex media contain one or more crude sources of nutrients and their exact chemical composition and
components are unknown;
• defined media, or synthetic media, are media in which the components of the medium are chemically
known and are present in relatively pure form.
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Requirements for growth of micro-organisms
Microorganisms require an energy source (which may be chemical or light) and simple chemical compounds for
biosynthesis.
Many microorganisms can produce all of the complex molecules required for life, including amino acids for protein
synthesis.
Other microorganisms require more complex compounds to be added to the growth media, including vitamins and
fatty acids.
Environmental conditions
In order to grow cells in culture, they must be supplied with a growth medium and the correct environmental
conditions, including:

sterility to eliminate contamination e.g. using aseptic techniques; heat sterilisation by autoclaving

temperature - controlled using an incubator

pH - controlled by the use of buffers or addition of acid/alkali

gaseous environment - some microorganisms are anaerobic and will not grow in the presence of
oxygen; others will require a good oxygen supply

light (if it is a photosynthetic microorganism)

Industrial production methods
Production of micro-organisms on an industrial scale requires the use of large fermenters that are computercontrolled. Culture conditions are monitored by sensors and conditions are maintained at optimum levels.
Patterns of growth
Growth is the irreversible increase in dry biomass. Growth occurs when rate of synthesis of organic materials
exceeds rate of breakdown. Measuring dry biomass is not always practicable: investigation of bacterial growth
involves measuring the increase in cell number over time.
The time taken for a cell to divide into two cells (to double) is called generation time.
Under ideal conditions, some species of bacteria are capable of doubling in number every 20 minutes.
The four main stages in growth are:
1. Lag phase - micro-organisms adjust to the conditions of
the culture by producing enzymes that metabolise the
available substrates.
2. Exponential or log (logarithmic) phase - rate of growth
is at its highest
3. Stationary phase - culture medium becomes depleted
and secondary metabolites are produced.
4. Death phase - lack of substrate and toxic accumulation
of metabolites causes death of cells
If bacterial growth during the exponential (log) phase is
plotted on normal graph paper, there is not enough space
on the y axis, or the scale is so reduced that it makes
plotting or reading with accuracy almost impossible.
The solution is to use semi-logarithmic graph paper. This
has been printed in a specific way to allow data which has
a very wide range to be plotted.
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Primary and Secondary metabolism
Filamentous fungi (and some bacteria) exhibit two types of metabolism: Primary and Secondary.
Primary metabolism
Metabolism during lag and exponential (log) phases of growth: breakdown of substrate and production of primary
metabolites e.g. amino acids and nucleotides for synthesis of microbial cells.
Primary metabolites also include end products of fermentation e.g. ethanol, a waste product but one of the first
microbial metabolites used by humans.
Secondary metabolism
Metabolism at end of log phase and during entire stationary phase: production of secondary metabolites that are
not used for growth (some may be toxic) but may confer an advantage e.g. antibiotics inhibit bacteria.
Humans grow micro-organisms on an industrial scale in order to harvest many secondary metabolites e.g.:
 Antibiotics
 Cyclosporin (immunosuppressant)
 Gibberellin (plant hormone)
 Glutamic acid (amino acid used to make monosodium glutamate)
 Citric acid
Manipulation of metabolism
Micro-organisms naturally control their metabolic pathways by several processes e.g.
 induction or inhibition of enzymes;
 end-product inhibition.
In industrial processes, micro-organisms are used to produce large amounts of a desired compound. This involves
manipulation of microbial metabolism as the desired compound is often an intermediate of a metabolic pathway
which, under normal circumstances, would not accumulate in large quantities.
The production of these useful substances is often stimulated by the addition of precursors, inducers or inhibitors.
Precursor: an earlier metabolite in the pathway.
Inducer: a metabolite that induces the formation of an enzyme.
Inhibitor: reduces the activity of an enzyme by acting competitively or non-competitively or by binding to the
operator region of the coding gene.
Microbial metabolic pathway:
In this metabolic pathway, metabolite C is the desired
product.
C can be mass-produced by adding:
 a precursor - in this case metabolite A, providing
a continuous supply of metabolite B;
 an inducer – such as metabolite B – that would
induce the formation enzyme 2;
 an inhibitor of enzyme 3, allowing metabolite C
to accumulate.
•
•
•
•
•
•
Example of industrial process: production of penicillin
Spores of Penicillium chrysogenum germinated.
Mycelium used to inoculate liquid medium in fermenter.
Optimum conditions promote rapid growth – vegetative phase.
Balance of nutrients altered: slows down growth and increases antibiotic production – antibiotic
production phase.
Fungal mycelium filtered out.
Penicillin recovered by series of chemical processes giving 99.5% pure crystals of penicillin.
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GENETIC CONTROL OF METABOLISM IN MICRO-ORGANISMS
Use of micro-organisms
Wild strains of micro-organisms have been used in industry for many years. These wild strains can be cultivated so
that:
• they produce required substances more efficiently;
• useful mutant strains can be isolated and cultivated.
Wild mutants may need to be improved in order to confer e.g.
• genetic stability
• ability to grow on low-cost nutrients
• ability to overproduce required product
• ease of harvesting
Strain improvement
Strain improvement - to alter a wild strain’s genome - can be brought about by:
• mutagenesis
• selective breeding
• recombinant DNA technology
Mutagenesis
Mutations occur naturally but can also be induced artificially by mutagenesis.
Mutagenesis is caused by the use of mutagenic agents such as radiation and mutagenic chemicals.
Most mutations are deleterious but some may produce an improved strain which is beneficial to the organism or
makes it more useful to humans.
Many micro-organisms used in industry have been improved by repeated mutagenesis and selection for the
desired trait. The improved strain usually lacks an inhibitory control mechanism, allowing it to produce more of the
required end-product.
Mutant strains are often genetically unstable: they regress to the wild type after generations in culture by
undergoing a reverse mutation. Improved strains must be monitored to ensure that this has not occurred.
Selective breeding
Eukaryotic micro-organisms can reproduce sexually and asexually. Sexual reproduction in fungi leads to variation
and can be used in breeding programmes to produce new strains.
Bacterial reproduction is asexual but horizontal transfer of genetic material can lead to variation and new strains.
Bacteria can pass plasmids or pieces of chromosomal DNA to each other in this way, leading to variation when
different strains are cultured together.
Horizontal transfer can occur in several ways:
• transformation
• transduction
• conjugation
Transformation
Bacterial cell takes up a piece of DNA from the
remains of another bacterium and
incorporates it into its own DNA.
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Transduction
Foreign DNA (from another bacterial cell) is introduced
by a bacteriophage and incorporated into host
bacterium’s DNA.
Notes
Conjugation
Conjugation tube formed between two different
bacterial cells allows transfer of replicated plasmid.
Transduction
Recombinant DNA technology
Recombinant DNA technology is a technique used in genetic engineering where a required plant or animal gene
sequence is transferred into a micro-organism.
The micro-organism has been artificially transformed so that it will:
• produce more of a target product;
• secrete the product into the surrounding medium;
• not survive in an external environment (safety mechanism).
Gene sequences are transferred from donor cells to the host bacterium using a vector such as a plasmid.
The transformed host will express the new gene, producing e.g. insulin. The host cell has recombinant DNA - a
combination of its own and foreign DNA.
Recombinant DNA technology techniques
Recombinant DNA technology requires:
• donor cells with required gene;
• restriction endonucleases to cut up DNA;
• vector e.g. plasmid to carry DNA from donor to host;
• DNA ligase to seal DNA fragment into plasmid
• host cells to receive the altered vector.
Restriction endonucleases
Restriction endonucleases are a group of enzymes that can recognise and cut specific sequences of DNA into
fragments with ’sticky ends’ (pieces of DNA that have unpaired nucleotides at each end).
They allow specific genes to be cut out of a source chromosome as well as the cutting of bacterial plasmids.
Each endonuclease recognises a different restriction site (DNA sequence 4-8 nucleotides long).
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Vectors
To be effective, a vector must have:
• restriction site that can be cut open by the same restriction endonuclease used to the cut the donor DNA;
• marker gene to indicate whether host cell has taken up the vector e.g. gene for penicillin resistance (cells
that have no marker are killed by penicillin);
• origin of replication controlling self-replication of plasmid and regulatory sequences controlling gene
expression.
DNA ligase
DNA ligase is an enzyme that can join two different fragments of DNA together. It seals DNA fragments into a
plasmid to form a recombinant plasmid. The sticky ends of the DNA fragments and the plasmids are complementary
to each other, allowing ligase to bind them together.
Steps in the process
• • Identification of gene of interest in donor
• Isolation of gene of interest
• Insertion of gene into vector
• Introduction of vector to host (transformation).
• Selection of transformed host cells
• Expression of introduced gene in host
Artificial chromosomes
Artificial chromosomes, constructed by scientists, can also act as vectors. They are usually made by adding nonbacterial DNA to bacterial chromosomes. They are useful in that they carry larger fragments of DNA than is possible
using plasmids.
Use of bacteria – limitations
Eukaryote DNA
Prokaryote DNA
• introns and exons
• exons but no introns
• primary transcripts modified by splicing
• no modification by splicing
• proteins undergo post-translational modification.
• no post-translational modification
These differences may lead to the production of an inactive polypeptide due to incorrect folding or lack of posttranslational modification. Some proteins are therefore more successfully produced by using genetically
transformed eukaryotic cells e.g. yeast.
Hazards and control of risks
Requirements for licence
Product must be:
• safe for manufacturing staff
• safe for consumers
• pure
• uncontaminated by micro-organisms
• fit for purpose
Manufacturing process must:
• maintain standards of product purity
• use safe, well-designed facilities
Risk assessment
Many micro-organisms used in biotechnology can act as
allergens, irritants or pathogens and their use requires
stringent risk assessment.
A risk assessment must:
1. assess the risk
2. identify potential hazards
3. construct and apply control measures
4. review effectiveness and adopt improvements
5. regularly repeat the process
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Higher Biology
Metabolism & Survival
Notes
Ethical considerations
Ethics can be defined as ‘moral principles that govern a person's behaviour or the conducting of an activity ‘(Oxford
dictionary). Regarding recombinant DNA technology, it deals with issues of what is right and wrong in research and
development in this field.
There are many arguments on both sides.
Arguments in favour
Recombinant DNA technology leads to improvement of:
• nutrition and food security (more and better food)
• the environment, by reducing use of pesticides and fertilisers
• health, by efficient drug production
Arguments against
Recombinant DNA technology could lead to:
• uncontrolled dispersal of altered cells into the natural environment;
• sideways transfer of genetic material to different species;
• unforeseen and difficult to control metabolic modifications;
• creation of new pathogenic micro-organisms.
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