WATER AND BIOLOGICAL MOLECULES
BEST ‘A’ LEVEL BIOLOGY NOTES
ENZYMES
TUTOR: Taruvinga G
DIP ED. HBScED.
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BEST ‘A’ LEVEL BIOLOGY NOTES compiled by TARUVINGA G   0772 980 253 Page 1
WATER AND BIOLOGICAL MOLECULES
ENZYMES
KEY
CONCEPT
OBJECTIVES
Learners should be
able to:
8.2.3
1. explain the mode
Enzymes
of action of
enzymes
CONTENT
(ATTITUDES, SKILLS
AND KNOWLEDGE)
- Lock and key
hypothesis
- Induced fit
hypothesis
2. follow the
- Enzyme catalyzed
progress of an
reactions
enzyme catalyzed - Effects of
reaction
temperature, pH,
enzyme
3. explain factors
concentration and
affecting rate of
substrate
enzyme catalysed concentration
reactions
- Reversible and
non- reversible
4. explain the effect
inhibition
of competitive
- Inhibitors such as
and non –
heavy metals
competitive
(cyanide, mercury),
inhibitors on
insecticides
enzyme activity
SUGGESTED LEARNING
ACTIVITIES AND NOTES
SUGGESTED
RESOURCES
 Biuret reagents
 ICT tools
 Braille
software/Jaws
- Measuring the rate of
 Print media
formation of products or  Models
rates of disappearance
(buttons/beads
of substrates.
threads)
 Catalase
- Carrying out
 Amylase
experiments to show
 Substrates
effects of the factors on
 Buffers
the rate of reactions.
 Acids and bases
 Inhibitors
- Demonstrating effects
of inhibitors on enzyme  Models of
enzymes
catalysed reactions.
- Constructing models to
demonstrate the mode
of action of enzymes.
ENZYMES
— Enzymes are globular proteins that serve as biological catalysts.
— They speed up or slow down metabolic reaction, but remain unchanged.
— They may facilitate the breaking of an existing bond or the formation of a new bond.
— Substrates = the molecules that bind to the enzyme
— Products = new substances formed.
1. Active sites
— Active site = area in enzyme's molecule where the substrate bind to enzyme.
— Enzyme substrate complex formed when enzyme‟s active site binds with substrate.
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 The R groups of amino acids at the active site form temporary bonds with the
substrate molecule. This pulls the substrate slightly out of shape, causing it to react
and form products.
2. Activation energy
Activation energy = energy the substrates need for changing themselves into
products. Heating provides activation energy.
Enzymes reduce activation energy needed ---> reaction take place at low to. They do
this by distorting the shape of the substrate when it binds at the enzyme's active
site.
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3. Enzyme specificity
Lock and Key hypothesis (Emil Fisher, 1894)
— The shape of the active site of the enzyme and the substrate molecules are
complementary.
— They possess specific 3-D shapes that fit exactly into one another.
— Like a key into a lock, only the correct size and shape of the substrate (the key)
would fit into the active site of the enzyme (the lock).
— This shows the high specificity of enzymes, however it is too rigid.
The active site has a complementary shape to the substrate.
Induced fit hypothesis (Koshland, 1958)
— The shape of the active site of the enzyme and the substrate molecules are NOT
complementary.
— In the presence of the substrate, the active site continually reshapes by its
interactions with the substrate, until the substrate is completely fit into it.
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— The enzyme is flexible and molds to fit the substrate molecule like gloves fitting
one„s hand or clothing on a person.
— This hypothesis is more acceptable.
The active site forms a complementary shape to the substrate only after binding.
Following the course of an enzyme-catalysed reaction
Measurement of the rate of formation of the product or the rate of disappearance of
the substrate.
1. Measurement of the rate of formation of O2 in the reaction:
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— Mash up some biological material like potato tuber or celery stalks, mix them with
water and filter the mixture to obtain a solution containing catalases.
— Add the mixture to H2O2 (hydrogen peroxide) in a test tube. Use small tubes --> not
too much gas in the tube above the liquid.
— Collect the gas in a gas syringe and recording the volume every minute until the
reaction stops.
Note - You can replace the gas syringe by an inverted measuring cylinder over water.
2. Measurement of the rate of disappearance of starch in the reaction:
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WATER AND BIOLOGICAL MOLECULES
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Add amylase solution to starch suspension in a test-tube.
Take samples of the reacting mixture at regular time intervals, and test for the
presence of starch using iodine in KI solution.
When starch is present, iodine is dark blue.
If the blue colour lightens, starch is breaking down.
When there is no starch, the iodine solution will remain orange-brown.
FACTORS AFFECTING THE RATE OF ENZYME-CATALYSED REACTIONS
These factors are:
 Temperature
 pH
 Enzyme concentration
 Substrate concentration
 Inhibitor concentration
 Covalent modification
 Allosteric effectors
When an enzyme solution is added to a solution of its substrate, the molecules
collide.
With time, the quantity of substrate decreases because it is being changed into product.
This means frequency of collisions decreases and rate of reaction gradually decreases.
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The reaction rate is fastest at the start of the reaction when substrate concentration is
greatest. Therefore, when comparing reaction rates of an enzyme in different
circumstances, we should measure the initial rate of reaction.
1. Temperature
— As to increases, kinetic energy of reacting molecules increases. Collision frequency
increases causing an increase in the rate of reaction.
 Up to the optimum temperature the rate increases geometrically with
temperature (i.e. it's a curve, not a straight line). The rate increases because the
enzyme and substrate molecules both have more kinetic energy so collide more
often, and also because more molecules have sufficient energy to overcome the
(greatly reduced) activation energy.
 The increase in rate with temperature can be quantified as a Q10, which is the
relative increase for a 10°C rise in temperature.
 Q10 is calculated as follows:
Q10 is usually 2-3 for enzyme-catalysed reactions (i.e. the rate doubles every
10°C) and usually less than 2 for non-enzyme reactions.
— At optimal to enzyme's activity is maximal and rate is maximal.
— Above this to, hydrogen bonds holding enzyme molecule in shape begin to break.
This cause a change in tertiary structure of the enzyme, an effect called denaturation.
The active site is deformed, so less binding of substrate with enzyme occurs,
resulting in decreased rate of reaction.
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 Enzymes have an optimum temperature at which they work fastest. For mammalian
enzymes this is about 40°C, but there are enzymes that work best at very different
temperatures, e.g. enzymes from the arctic snow flea work at -10°C, and enzymes
from thermophilic bacteria work at 90°C.
o
 Enzymes in the human body generally have an optimum to of about 37 C, organisms
that have evolved to live in much higher or lower temperatures may have much
higher or lower optimum to.
 The rate of enzyme reaction is not zero at 0°C, so enzymes still work in the fridge
(and food still goes off), but they work slowly. Enzymes can even work in ice, though
the rate is extremely slow due to the very slow diffusion of enzyme and substrate
molecules through the ice lattice.
2. pH
 Most enzyme molecules only maintain their correct tertiary structure (exhibit
maximum activity) within a very narrow pH range.
 Optimum pH - is the pH at which an enzyme has maximum activity. Biological
buffers help maintain the optimum pH for an enzyme.
 Changes in pH can make and break intra- and intermolecular bonds, changing the
shape of the enzyme and, therefore, its effectiveness.
 Most enzymes have an optimum pH that falls within the physiological range of 7.07.5.
 Notable exceptions are the digestive enzymes pepsin and trysin:
pepsin (active in the stomach) - optimum pH of 1.5
trypsin (active in the small intestine) - optimum pH of 8.0.
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3. Enzyme concentration
When there is more substrate than enzyme:
— limiting factor is Enzyme concentration
— ↑ concentration of enzyme --> ↑ collisions between enzyme and substrate -->↑ rate
of the reaction (directly proportional to the enzyme concentration )
— Increasing the enzyme concentration beyond a certain point does not change the
rate of reaction BECAUSE when there are less substrate than enzyme:
 limiting factor is Substrate concentration
 ↑ concentration of enzyme does NOT ↑ rate of reaction.
Limiting factor:
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Factor that directly affects the rate of reaction at which a process occurs if its quantity is
changed.
Its value has to be ↑ in order to ↑ the rate.
4. Substrate concentration
 The rate of reaction and substrate concentration are directly proportional, up to a
given point.
 This point is called Vmax,
 At Vmax, all the enzyme active sites are said to be „saturated‟ or occupied.
 Increasing the substrate concentration beyond this point does not change the rate
of reaction BECAUSE there are no available active sites free to bind with
substrates.
 At Vmax, the amount of enzyme becomes the limiting factor.
5. Inhibitor concentration
Inhibitor = a substance that slows down the rate at which an enzyme works.
Competitive inhibitors
— A competitive inhibitor molecule has a similar structure to the normal substrate
molecule, and it can fit into the active site of the enzyme, preventing the substrate
from binding.
— It therefore competes with the substrate for the active site, so the reaction is
slower.
— The greater the proportion inhibitor: substrate, the more inhibitor molecules (not
substrate molecules) will bump into an active site.
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— Relative concentrations of the inhibitor and the substrate will affect the degree
to which a competitive inhibitor slows down a reaction.
 Effect is concentration dependent
 Can be minimized by raising the concentration of substrate
 Effects are reversible
 Examples of competitive inhibitors
 Example 1: an individual who swallows methanol is in danger of becoming
blind. This is because the methanol – which itself is not toxic – will be
metabolized to formaldehyde which is extremely toxic and will cause blindness.
At the hospital, the individual will be treated with ethanol. The ethanol is
structurally similar to methanol and will compete with methanol for the enzyme’s
active sites. Thus, the metabolism of methanol is slowed down.
 Example 2: arabinose competes with glucose for the active sites on glucose
oxidase.
 Example 3: oxaloacetate, malonate and pyrophosphate all compete with
succinate for the active site of the enzyme succinate dehydrogenase.
Non-competitive inhibitors
— Non-competitive inhibitor molecule has different shape from the substrate molecule
and does not fit into the active site. It binds to another part of the enzyme molecule
called the allosteric site, changing the shape of the whole enzyme, including the
active site, so that it can no longer bind substrate molecules.
— This is permanent and cannot be rectified
— Effect is independent of substrate concentration
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Some poisons act as metabolic inhibitors. For example, potassium cyanide is a noncompetitive inhibitor to the enzyme cytochrome oxidase. The effect of this poison is to
decrease the body’s ability to respire using oxygen, so it begins to respire anaerobically,
causing lactic acid accumulation in the blood. Potassium cyanide is used in the
extraction of gold. Animals/livestock that accidentally drink the waste water from a gold
processing plant will die due to fast accumulation of lactic acid in their body tissues.
Reversible inhibitor
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Bind reversibly to the enzyme
Have no long-lasting effect since they detach themselves after a while
Irreversible inhibitor
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Binds irreversibly
Even if in high competition with substrate, can eventually inhibit a large
proportion of available enzyme
Can have long-lasting effects
Until organism generates more enzyme and the inhibitor is outnumbered by
enzyme
 For example,
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Aspirin is an irreversible inhibitor of cycloxygenase, an enzyme
involved in the synthesis of prostaglandins.
 Substances such as mercury, iron and arsenic bind irreversibly to the
SH (sulphydryl) group on enzymes.
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6. Covalent modification
The activity of some enzymes is controlled by other enzymes, which modify the protein
chain by cutting it, or adding a phosphate or methyl group. This modification can turn
an inactive enzyme into an active enzyme (or vice versa), and this is used to control
many metabolic enzymes and to switch on enzymes in the gut e.g. hydrochloric acid in
stomach activates pepsin which activates rennin.
7. Allosteric Effectors
The activity of some enzymes is controlled by certain molecules binding to a specific
regulatory (or allosteric) site on the enzyme, distinct from the active site. Different
molecules can inhibit or activate the enzyme, allowing sophisticated control of the rate.
Only a few enzymes can do this, and they are often at the start of a long biochemical
pathway. They are generally activated by the substrate of the pathway and inhibited by
the product of the pathway, thus only turning the pathway on when it is needed.
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Coenzymes, cofactors & prosthetic groups
Cofactor = non-protein component of an enzyme that is required in order for the
enzyme to function.
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E.g. Cl – is a co-factor for amylase
Coenzyme = organic, non-protein molecule whose role is to transport chemical groups
between enzymes, linking together controlled enzyme reactions.
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E.g. Vitamins are a diverse group of coenzymes
NADP is a coenzyme to photosynthesis
NAD and FAD are coenzymes in respiration
Prosthetic group = coenzyme that is a permanent part of the enzyme

E.g. Zn2+ is a prosthetic group in carbonic anhydrase
INVESTIGATING FACTORS AFFECTING THE RATE OF ENZYME-CATALYSED
REACTIONS
1. Temperature
The effect of to on enzyme activity
You can use almost any enzyme reaction for this, such as the action of catalase on
H2O2.
 Set up several conical flasks containing H2O2 (the same volume and
concentration).
 Stand each one in a water bath at a particular to. Use at least 5 to over a good
range (0-90 oC). Better to set up 3 sets of tubes at each to --> mean result for
each to.
 Take a set of test tubes and add the same volume of catalase solution to each
one.
 Stand these in the same set of water baths.
 Leave all the flasks and tubes to come to the correct to. Check with a
thermometer.
 Take the first flask with H2O2, dry its base and sides and stand it on a sensitive
toppan balance. Pour in the catalase solution (same to) and immediately take the
balance reading.
 Record the new balance readings every 30 seconds for about 3 minutes. The
readings will ↓ as O2 is given off.
 Repeat with the solutions kept at other temperatures.
 Work out the initial rate of each reaction, either taken directly from your readings,
or by drawing a graph of mass lost (mass of O2) against time for each to, and
then working out the gradient of the graph over the first 30 seconds or 60
seconds of the reaction.
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Use your results to plot a graph of initial rate of reaction (y-axis) against to.
2. pH
The effect of pH on enzyme activity
Investigating the effect of to on the rate of breakdown of H2O2 by catalase.
Vary pH by using different buffer solutions added to each enzyme solution.
o
Keep t , enzyme concentration, substrate concentration and total volume of reactants
the same for all the tubes.
Record, process and display results.
3. Substrate concentration
The effect of substrate concentration on enzyme activity
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You can do this in the same way as described for investigating the effect of enzyme
concentration, but this time keep the concentration of catalase the same and vary the
concentration of hydrogen peroxide.
4. Enzyme concentration
The effect of enzyme concentration on rate of reaction
You could use the following method to investigate the effect of enzyme concentration on
the rate at which the enzyme catalase converts its substrate H2O2 to H2O and O2 .
 Prepare a catalase solution
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Prepare different dilutions of this solution: and so on. The final 'solution' prepared
should be 10 cm3 of distilled water.
Place each solution into a tube fitted with a gas syringe. Use small tubes -->
there is not too much gas in the tube above the liquid, but leave space to add an
equal volume of H2O2 solution at the next step. Label tube with a waterproof
marker. Better to prepare 3 sets of these solutions.
Place each tube in a water bath at 30oC.
Take another set of tubes and add 10 cm3 of H2O2 solution to each one.
The concentration of H2O2 must be the same in each tube. Stand these tubes in
the same water bath.
Leave all the tubes for 5 minutes --> correct to. Add the contents of 1 of the H2O2
tubes to the first enzyme tube. Mix thoroughly.
Measure the volume of gas collected in the gas syringe after 2 minutes. If you
are using 3 sets, then repeat using the other 2 tubes containing the same
concentration of enzyme.
Do the same for each of the tubes of enzyme. Record the mean volume of gas
produced in 2 minutes for each enzyme concentration and plot a line graph to
display your results.
Note: if you find that you get measurable volumes of gas sooner than
2 minutes after mixing the enzyme and substrate --> take readings earlier.
The closer to the start of the reaction you make the measurements, the better.
reaction you make the measurements, the better.
SUMMARY NOTES: ENZYMES
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Enzyme action
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All enzymes are globular proteins → spherical in shape
Control biochemical reactions in cells
They have the suffix "-ase"
Intracellular enzymes are found inside the cell
Extracellular enzymes act outside the cell
Enzymes are catalysts → speed up chemical reactions
Reduce activation energy required to start a reaction between molecules
Substrates (reactants) are converted into products
Reaction may not take place in absence of enzymes (each enzyme has a specific
catalytic action)
Enzymes catalyse a reaction at maximum rate at an optimum state.
The substrate and the enzyme must collide with sufficient energy.
Enzymes work by lowering the activation energy required to start a reaction.
Once the substrate is inside the active site, the enzyme changes shape slightly,
distorting the molecule in the active site, and making it more likely to change into the
product.
It's a bit more complicated than that though. Although enzymes can change the
speed of a chemical reaction, they cannot change its direction, otherwise they could
make "impossible" reactions happen and break the laws of thermodynamics.
When a substrate (or product) molecule binds, the active site changes shape and fits
itself around the molecule, distorting it into forming the transition state, and so
speeding up the reaction. This is sometimes called the induced fit mechanism.
Factors affecting enzyme action
Measuring enzyme-catalysed reactions
 To measure the progress of an enzyme-catalysed reaction, its time course is
measured. This is how long it takes to run its course.
 The two “events” most frequently measured are the volume of gas produced during a
reaction and the disappearance of a substrate.
Effect of Temperature
 Enzymes have an optimum temperature at which they work fastest. For mammalian
enzymes this is about 40°C, but there are enzymes that work best at very different
temperatures, e.g. enzymes from the arctic snow flea work at -10°C, and enzymes
from thermophilic bacteria work at 90°C.
 The rate of reaction doubles, approximately almost every ten degrees.
 The rate of reaction will increase as temperature increases. Then, once it reaches its
optimum temperature it will begin to decrease as the temperature rises due to the
active site being denatured.
 The thermal energy breaks the hydrogen bonds holding the secondary and tertiary
structure of the enzyme together, so the enzyme (and especially the active site) loses
its shape to become a random coil.
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Effect of pH
 Enzymes have an optimum pH at which they work fastest.
 For most enzymes this is about pH 7-8 (physiological pH of most cells), but a few
enzymes can work at extreme pH, such as protease enzymes in animal stomachs,
which have an optimum of pH 1.
 The pH affects the charge of the amino acids at the active site, so the properties of
the active site change and the substrate can no longer bind. For example a carboxyl
acid R groups will be uncharged a low pH (COOH), but charged at high pH (COO ).
ENZYME INHIBITION
 Inhibitors inhibit the activity of enzymes, reducing the rate of reactions. They are
found naturally, but are also used artificially as drugs, pesticides and research tools.
There are two kinds of inhibitors, competitive and non-competitive.
Competitive inhibitor
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— A competitive inhibitor molecule has a similar structure to the normal substrate
molecule, and it can fit into the active site of the enzyme.
— It therefore competes with the substrate for the active site, so the reaction is slower.
— It is the difference between the concentration of the inhibitor and the concentration of
the substrate that determines the affect it has on the enzymes activity.
— The inhibitor is not permanently bonded to the active site so once it leaves a
substrate molecule can take its place.
— Eventually all the substrate molecules will be in the active sites. However, depending
on the concentration of the inhibitor, the longer this will take.
Non-competitive inhibitors
— Non-competitive inhibitors do not fit into the active site but instead they bind to
another part of the enzyme molecule, changing the shape of the whole enzyme,
including the active site, so that it can no longer bind substrate molecules.
— Inhibitors that bind fairly weakly and can be washed out are sometimes called
reversible inhibitors, while those that bind tightly and cannot be washed out are
called irreversible inhibitors. Poisons like cyanide, heavy metal ions and some
insecticides are all non-competitive inhibitors.
— Non-competitive inhibitors therefore simply reduce the amount of active enzyme (just
like decreasing the enzyme concentration).
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