2022-24 syllabus By: Sultan ALShehri

Enzymes
Mode of action of enzymes
 Enzymes as Proteins
 Enzyme: a protein produced by a living organism that acts as a biological
catalyst in a chemical reaction by reducing activation energy
 Enzymes are biological catalysts
o ‘Biological’ because they function in living systems
o ‘Catalysts’ because they speed up the rate of chemical reactions without being used up or
changed
 Enzymes are also globular proteins, they fold up into precise shapes.
 Critical to the enzyme’s function is the active site where the substrate binds
 Metabolic pathways are controlled by enzymes in a biochemical cascade of
reactions
o Virtually every metabolic reaction within living organisms is catalysed by an enzyme –
enzymes are therefore essential for life to exist
 Many enzyme names end in –ase; for example, amylase and ATPase
 Enzymes can be intracellular or extracellular referring to whether they are
active inside or outside the cell respectively
 Intracellular enzymes are produced and function inside the cell
 Extracellular enzymes are secreted by cells and catalyse
reactions outside cells (eg. digestive enzymes in the gut)

 Don’t forget that enzymes are proteins and so anything that could denature a
protein, rendering it non-operational (extremes of heat, temperature, pH etc.)
would also denature an enzyme.
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 The mode of action of enzymes

 Enzymes have an active site which is an area where specific substrates
bind forming an enzyme-substrate complex
 The active site of an enzyme has a specific shape to fit a specific substrate
 Extremes of heat or pH can change the shape of the active site, preventing
substrate binding – this is called denaturation
 Substrates collide with the enzymes active site and this must happen at the
correct orientation and speed in order for a reaction to occur

The active site of an enzyme has a specific shape to fit a specific substrate (when the
substrate binds an enzyme-substrate complex is formed)
 The specificity of an enzyme is a result of the complementary nature between
the shape of the active site on the enzyme and its substrate(s)

 The shape of the active site (and therefore the specificity of the enzyme) is
determined by the complex tertiary structure of the protein that makes up the
enzyme:
o Proteins are formed from chains of amino acids held together by peptide bonds
o The order of amino acids determines the shape of an enzyme
o If the order is altered, the resulting three-dimensional shape changes

An example of enzyme specificity – the enzyme catalase can bind to its substrate hydrogen
peroxide as they are complementary in shape, whereas DNA polymerase is not
 An enzyme-substrate complex forms when an enzyme and its substrate join
together
 The enzyme-substrate complex is only formed temporarily, before the
enzyme catalyses the reaction and the product(s) are released

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The temporary formation of an enzyme-substrate complex

 Enzyme reactions can either be catabolic or anabolic
 Catabolic reactions involve the breakdown of complex molecules into simpler
products, which happens when a single substrate is drawn into the active site
and broken apart into two or more distinct molecules
 Examples of catabolic reactions include cellular respiration and
hydrolysis reactions

A catabolic reaction

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 Anabolic reactions involve the building of more complex molecules from

simpler ones by drawing two or more substrates into the active site, forming
bonds between them and releasing a single product
 Examples of anabolic reactions include protein synthesis and
photosynthesis

An anabolic reaction

 Enzymes work by lowering the activation energy of a reaction

 All chemical reactions are associated with energy changes
 For a reaction to proceed there must be enough activation energy
 Activation energy is the amount of energy needed by the substrate to become
just unstable enough for a reaction to occur and for products to be formed
o Enzymes speed up chemical reactions because they influence the stability of bonds in the
reactants
o The destabilisation of bonds in the substrate makes it more reactive
 Enzymes work by lowering the activation energy of a reaction and in doing so
they provide an alternative energy pathway

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The activation energy of a chemical reaction is lowered by the presence of a catalyst

(ie. an enzyme)
 Don’t forget that both enzymes and their substrates are highly specific to each
other – this is known as enzyme-substrate specificity.

 How Enzymes Work

a) The lock-and-key hypothesis
 Lock-and-key hypothesis: a hypothesis for enzyme action; the substrate is a
complementary shape to the active site of the enzyme, and fits exactly into
the site; the enzyme shows specificity for the substrate
 Enzymes are globular proteins
 This means their shape (as well as the shape of the active site of an enzyme) is
determined by the complex tertiary structure of the protein that makes up the
enzyme and is therefore highly specific
 In the 1890’s the first model of enzyme activity was described by Emil Fischer:
o He suggested that both enzymes and substrates were rigid structures that locked into each
other very precisely, much like a key going into a lock
o This is known as the ‘lock-and-key hypothesis’
 This was later modified and adapted to our current understanding of enzyme
activity, permitted by advances in techniques in the molecular sciences
 The whole idea can be summarized as follows:

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The lock-and-key hypothesis

b) The induced-fit hypothesis

 The modified model of enzyme activity is known as the ‘induced-fit
hypothesis’
 Induced-fit hypothesis: a hypothesis for enzyme action; the substrate is a
complementary shape to the active site of the enzyme, but not an exact fit –
the enzyme, or sometimes the substrate, can change shape slightly to ensure
a perfect fit, but it is still described as showing specificity
 Although it is very similar to the lock and key hypothesis, in this model the
enzyme and substrate interact with each other:
o The enzyme’s active site isn’t initially an exact fit to the substrate molecule, so the enzyme
(it’s more flexible) and its active site (and sometimes the substrate) can change shape slightly
as the substrate molecule enters the enzyme
o These changes in shape are known as conformational changes
o This ensures an ideal binding arrangement between the enzyme and substrate is achieved
o This maximises the ability of the enzyme to catalyse the reaction

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The induced-fit hypothesis

 Don’t forget – our current understanding of enzyme-substrate interactions is
based on the induced-fit hypothesis.

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 Investigating the progress of enzyme-catalysed reactions

 The progress of enzyme-catalysed reactions can be investigated by:
o Measuring the rate of formation of products using catalase
o Measuring the rate of disappearance of substrate using amylase

 Investigating catalase activity

 In this investigation, the rate of product formation is used to measure the rate
of an enzyme-controlled reaction:
o Hydrogen peroxide is a common but toxic by-product of metabolism
o This means it must be broken down quickly
o Catalase is an enzyme found in the cells of most organisms that breaks hydrogen peroxide
down into water and oxygen
o Hydrogen peroxide and catalase are combined and the volume of oxygen generated is
measured in a set time
o The rate of reaction can then be calculated (and a graph could be plotted:
(rate of reaction = Volume of product produced/time taken)

Experimental set-up for investigating the rate of formation of a product using catalase

 Investigating amylase activity

 In this investigation, the rate of substrate disappearance is used to compare
rates of reaction under different conditions:
o Amylase is a digestive enzyme that hydrolyses starch into maltose and glucose
o Amylase functions best at pH 7 and 37oC (all enzymes operate best under specific conditions)
o Amylase and starch are combined and this reaction mixture is then tested for starch at
regular time intervals
o This can be done by taking samples from the reaction mixture at each time interval and
adding each sample to some iodine in potassium iodide solution (starch forms a blue-
black colour with this solution; when starch breakdown is complete, colour will change to
orange-brown)
o In this way, the time taken for starch to be broken down can be measured
o

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o Plot a graph of time vs absorbance

o The investigation can be repeated under a variety of conditions (eg. by altering pH or
temperature) and the reaction rates can then be compared

Experimental set-up for investigating the rate of disappearance of a substrate using amylase

 Course of a reaction: Initially, there’s a large number of substrates and every

enzyme has a substrate in its active site. The rate at which the reaction occurs
depends only on how many enzymes there are and the speed at which the
enzyme can convert the substrate into product, release it, and then bind with
another substrate. However, overtime, there are fewer substrates to bind
with enzymes; the reaction gets slower, until it eventually stops. The rate of
an enzyme-controlled reaction is always fastest at the beginning.

 The use of a colorimeter for measuring the progress of enzyme-catalysed

reactions
 Colorimeter: an instrument that measures the colour of a solution by
measuring the absorption of different wavelengths of light
 A colorimeter is able to measure light absorbance (how much light is
absorbed) or light transmission (how much light passes through) a substance
 Colorimetry can be used in any enzyme-catalysed reaction that involves colour
change

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 As the colour breaks down the transmission increases or light absorption

decreases and this can be used to measure the rate of the reaction
 For example, a colorimeter can be used to follow the progress of a starch-
amylase catalysed reaction as the amylase breaks the starch down into
maltose
 This can be carried out as follows:
o Colorimeter calibration: this is an important step in a colorimetric investigation and in this
case a weak iodine solution can be used to calibrate the colorimeter as the end point (or
100% transmission)
o Preparation of a starch solution of known concentration (stock solution), from which a range
of concentrations are made using serial dilutions (method outlined in diagram below)
o Following calibration and switching on the red filter (to maximise the percentage transmission
or absorbance), the colorimeter is used to measure the percentage absorbance or
percentage transmission values
o A calibration graph is then plotted of starch concentration (X-axis) vs percentage absorbance
or percentage transmission (Y-axis)

Serial dilution of starch to make a range of concentrations

 Factors that affect enzyme action

 The effects of the factors on the rate of enzyme-catalysed reactions
 The rate of enzyme-catalysed reactions is affected by the following factors:
o Temperature
o pH (using buffer solutions)
o Enzyme concentration
o Substrate concentration
o Inhibitor concentration

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 Temperature
 Enzymes have a specific optimum temperature – the temperature at which
they catalyse a reaction at the maximum rate
 Lower temperatures either prevent reactions from proceeding or slow them
down:
o Molecules move relatively slow
o Lower frequency of successful collisions between substrate molecules and active site of
enzyme
o Less frequent enzyme-substrate complex formation
o Substrate and enzyme collide with less (kinetic) energy, making it less likely for bonds to be
formed or broken (stopping the reaction from occurring)
 Higher temperatures speed up reactions:
o Molecules move more quickly
o Higher frequency successful collisions between substrate molecules and active site of
enzyme
o More frequent enzyme-substrate complex formation
o Substrate and enzyme collide with more (kinetic) energy, making it more likely for bonds to
be formed or broken (allowing the reaction to occur)
 However, as temperatures continue to increase, the rate at which an enzyme
catalyses a reaction drops sharply, as the enzyme begins to denature:
o Bonds (eg. hydrogen bonds) holding the enzyme molecule in its precise shape start to break
o This causes the tertiary structure of the protein (ie. the enzyme) to change
o This permanently damages the active site, preventing the substrate from binding
o Denaturation occurs if the substrate can no longer bind
o Very few human enzymes can function at temperatures above 50°C
 This is because humans maintain a body temperature of about 37°C, therefore even
temperatures exceeding 40°C will cause the denaturation of enzymes
 High temperatures causes the hydrogen bonds between amino acids to break, changing the
conformation of the enzyme

The effect of temperature on the rate of an enzyme-catalysed reaction

 pH
 The pH is a measure of the concentration of hydrogen ions.
 All enzymes have an optimum pH or a pH at which they operate best
 Enzymes are denatured at extremes of pH
o Hydrogen and ionic bonds hold the tertiary structure of the protein (ie. the enzyme) together

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o Below and above the optimum pH of an enzyme, solutions with an excess of H+ ions (acidic
solutions) and OH– ions (alkaline solutions) can cause these bonds to break
o This alters the shape of the active site, which means enzyme-substrate complexes form less
easily
o Eventually, enzyme-substrate complexes can no longer form at all
o At this point, complete denaturation of the enzyme has occurred
 Where an enzyme functions can be an indicator of its optimal environment:
o Eg. pepsin is found in the stomach, an acidic environment at pH 2 (due to the presence
of hydrochloric acid in the stomach’s gastric juice)
o Pepsin’s optimum pH, not surprisingly, is pH 2

The effect of pH on the rate of an enzyme-catalysed reaction for three different enzymes
(each with a different optimum pH)
 When investigating the effect of pH on the rate of an enzyme-catalysed
reaction, you can use buffer solutions to measure the rate of reaction
at different pH values:
o Buffer solutions each have a specific pH
o Buffer solutions maintain this specific pH, even if the reaction taking place would otherwise
cause the pH of the reaction mixture to change
o A measured volume of the buffer solution is added to the reaction mixture
o This same volume (of each buffer solution being used) should be added for each pH value
that is being investigated

 Note: Temperature can both affect the speed at which molecules are moving
(and therefore the number of collisions between enzyme and substrate in a
given time) and can denature enzymes (at high temperatures). pH, however,
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does not affect collision rate but only disrupts the ability of the substrate to
bind with the enzyme, reducing the number of successful collisions until
eventually, the active site changes shape so much that no more successful
collisions can occur.

 Enzyme Concentration
 Enzyme concentration affects the rate of reaction
 The higher the enzyme concentration in a reaction mixture, the greater the
number of active sites available and the greater the likelihood of enzyme-
substrate complex formation
 As long as there is sufficient substrate available, the initial rate of
reaction increases linearly with enzyme concentration
 If the amount of substrate is limited, at a certain point any further increase in
enzyme concentration will not increase the reaction rate as the amount of
substrate becomes a limiting factor

The effect of enzyme concentration on the rate of an enzyme-catalysed reaction

 Substrate Concentration
 The greater the substrate concentration, the higher the rate of reaction:
o As the number of substrate molecules increases, the likelihood of enzyme-substrate complex
formation increases
o If the enzyme concentration remains fixed but the amount of substrate is increased past a
certain point, however, all available active sites eventually become saturated and any further
increase in substrate concentration will not increase the reaction rate
o When the active sites of the enzymes are all full, any substrate molecules that are added
have nowhere to bind in order to form an enzyme-substrate complex
 For this reason, in the graph below there is a linear increase in reaction rate as
substrate is added, which then plateaus when all active sites become
occupied

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The effect of substrate concentration on the rate of an enzyme-catalysed reaction

 If substrate concentration is continually increased but enzyme concentration is kept
constant, there eventually comes a point where every enzyme active site is working
continuously. At this point, the substrate molecules are effectively ‘queuing up’ for an
active site to become available.
 At this stage, the enzyme is working at its maximum possible rate, known as V max (V
stands for velocity).

 Inhibitor Concentration
 There are two types of inhibitors:
o Competitive inhibitors have a similar shape to that of the substrate molecules and
therefore compete with the substrate for the active site
o Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape
of the active site and therefore prevents the substrate from binding to it
 Both types of inhibitors slow down or stop enzyme activity
 Increasing the concentration of an inhibitor, therefore, reduces the rate of
reaction and eventually, if inhibitor concentration continues to be increased,
the reaction will stop completely
 For competitive inhibitors, countering the increase in inhibitor concentration
by increasing the substrate concentration can increase the rate of reaction
once more (more substrate molecules mean they are more likely to collide
with enzymes and form enzyme-substrate complexes)
 For non-competitive inhibitors, increasing the substrate concentration cannot
increase the rate of reaction once more, as the shape of the active site of the
enzyme remains changed and enzyme-substrate complexes are still unable to
form

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The effect of inhibitor concentration on the rate of an enzyme-catalysed reaction

 While a competitive inhibitor will lower the initial rate of reaction (by
occupying some of the available active sites), eventually the same amount of
product will be produced as would have been produced without the
competitive inhibitor (the maximal rate is not affected).
 Non-competitive inhibitors lower the initial rate of reaction and the maximal
rate of reaction (a lower amount of product is produced than would normally
be produced).

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 Maximum rate of reaction (Vmax) & the Michaelis-Menten Constant (Km)

 Substrate concentration affects the rate of catalysis in an enzyme-substrate
reaction
 When the substrate concentration is fixed (and enzyme concentration is kept
constant) the initial rate of reaction is fastest and as active sites become
engaged(binded with other substrates), the reaction rate falls
 The Michaelis-Menten model describes the kinetics of such enzyme catalysed
reactions
 In this model, two values are used to describe an enzyme catalysed reaction,
the maximal rate or maximal velocity (Vmax) and the Michaelis-Menten
constant (Km)
 These values are derived from the reaction rate at different substrate
concentrations

 The maximum rate of reaction (Vmax) is used to derive the Michaelis–Menten

constant (Km), which is used to compare the affinity of different enzymes for
their substrates
 Michaelis-Menten enzyme kinetics
 The Michaelis-Menten model is used to investigate the kinetics of enzyme
catalysed reactions (enzyme kinetics is an area in biochemistry that studies
how different variables affect reaction rates)
 The rate of reaction is measured at different substrate concentrations,
producing a graph like the one below
 The two important values deduced are the Vmax (maximum rate of reaction at
saturating substrate concentrations) and the Km, which is the substrate
concentration at ½Vmax (also known as the Michaelis-Menten constant)
o The Michaelis-Menten constant is the substrate concentration at which the enzyme works
at half its maximum rate
o At this point, half of the active sites of the enzyme are occupied by substrate molecules
o The higher the affinity of the enzyme for the substrate, the lower the substrate concentration
needed for this to occur. Affinity is a measure of the strength of attraction between two
things. A high affinity means there is a strong attraction
o This is why the Michaelis-Menten constant is a measure of the affinity of an enzyme for its
substrate
 There is an inverse relationship between the Km and the affinity of an enzyme
for its substrate
 An enzyme with a high Km has a low affinity for its substrate and an enzyme
with a low Km has a high affinity for its substrate
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A graph showing the effect of substrate concentration on initial reaction rate, with
Vmax, ½Vmaxand Km values shown

 Reversible enzyme Inhibitors

 An enzyme’s activity can be reduced or stopped, temporarily, by a reversible

inhibitor
 There are two types of reversible inhibitors:
o Competitive inhibitors have a similar shape to that of the substrate molecules and
therefore compete with the substrate for the active site
o Non-competitive inhibitors bind to the enzyme at an alternative site, which alters the shape
of the active site and therefore prevents the substrate from binding to it

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Competitive and non-competitive inhibition

 Reversible inhibitors can act as regulators in metabolic pathways
 Metabolic reactions must be very tightly controlled and balanced, so that no
single enzyme can ‘run wild’ and continuously and uncontrollably generate
more and more of a particular product
 Metabolic reactions can be controlled by using the end-product of a particular
sequence of metabolic reactions as a non-competitive, reversible inhibitor:
o As the enzyme converts substrate to product, the process is itself slowed down as the end-
product of the reaction chain binds to an alternative site on the original enzyme, changing the
shape of the active site and preventing the formation of further enzyme-substrate complexes
o The end-product can then detach from the enzyme and be used elsewhere, allowing the
active site to reform and the enzyme to return to an active state
o This means that as product levels fall, the enzyme begins catalysing the reaction once again,
in a continuous feedback loop
o This process is known as end-product inhibition

End-product inhibition

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 A and C both have the same Vmax but at different time. They have different Km;
Higher Km when (competitive) Inhibitors are present.
 A and B have different Vmax but same Km

 The difference in activity between an immobilised enzyme and the same

enzyme free in solution
 Immobilised enzymes: enzymes that have been fixed to a surface or trapped
inside beads of agar gel
 Enzymes can be added to solutions and are thereby considered ‘free’ or they
can be immobilised
 Immobilised enzymes are enzymes that have been bound to an inert,
stationary and insoluble material such as alginate
 The substrate is then passed over the immobilised enzyme and the product is
collected
 Advantages to this method (immobilised enzyme):
o There is no enzyme in the product (the product is uncontaminated) and therefore there is no
need to further process or filter the end product
o The immobilised enzyme can be reused multiple times which is both efficient and cost-
effective (enzymes are expensive)
o Immobilised enzymes have a greater tolerance of temperature and pH
changes(immobilisation often makes enzymes more stable)
 Disadvantages:
o Active site may be distorted by immobilizing
o Substrate passes through matrix when immobilized
o Some product is retained within matrix
 A practical application of immobilised enzymes used in the food industry is in
the production of lactose-free milk:
o Milk is a valuable source of nutrients containing protein, fat and the carbohydrate Lactose
o 5-10% of the UK population are lactose intolerant
o Lactose is a disaccharide that is broken down into glucose and galactose

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Using immobilised enzyme to modify milk

 Using lactase as shown above is an efficient way to remove lactose from
milk and to provide lactose intolerant individuals with a way of consuming
milk without suffering intolerance symptoms:
o The enzyme lactase can be immobilised using alginate beads
o Milk is run through the column of lactase-containing beads
o The lactase hydrolyses the lactose in the milk to glucose and galactose
o This ensures the milk is lactose-free
o It can also then be used to make other lactose-free dairy products

Lactose is a disaccharide that is broken down by lactase into the monosaccharides

glucose and galactose

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