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CIE A Level Biology Your notes
3.1 Mode of Action of Enzymes
Contents
Enzymes
Enzyme Action
How Enzymes Work
Measuring Enzyme Activity
Colorimetry
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Enzymes
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Enzymes as Proteins
Enzymes are biological catalysts
‘Biological’ because they function in living systems
‘Catalysts’ because they speed up the rate of chemical reactions without being used up or
changed
Enzymes are also globular proteins
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
Virtually every metabolic reaction within living organisms is catalysed by an enzyme – enzymes are
therefore essential for life to exist
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 (e.g. digestive
enzymes in the gut)
Enzymes Table
Intracellular Extracellular
Example Catalase Amylase
Digestion of food is carried
Hydrogen peroxide is
out by extracellular
produced as a by-
enzymes
product of many cellular
This is because
reactions
macromolecules of food
Function It is harmful to cells
are too large to enter cells
Catalase converts
Amylase is an an example of
hydrogen peroxide into
a digestive enzyme
water and oxygen,
It digests carbohydrates
preventing cell damage
into simple sugars
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Exam Tip
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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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Enzyme Action
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Mode of Enzyme Action
Enzymes have an active site 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 enzyme's active site and this must happen at the correct orientation and
speed in order for a reaction to occur
Active Site Diagram
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:
Proteins are formed from chains of amino acids held together by peptide bonds
The order of amino acids determines the shape of an enzyme
If the order is altered, the resulting three-dimensional shape changes and therefore the enzyme's
shape and active site will change
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Enzyme Specificity Diagram
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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
Enzyme-Substrate Complex Diagram
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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
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A catabolic reaction
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
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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
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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
Enzymes speed up chemical reactions because they influence the stability of bonds in the Your notes
reactants
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
Enzyme Activation Graph
The activation energy of a chemical reaction is lowered by the presence of a catalyst (ie. an enzyme)
Exam Tip
Don't forget that both enzymes and their substrates are highly specific to each other – this is known as
enzyme-substrate specificity.
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How Enzymes Work
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How Enzymes Work
The lock-and-key hypothesis
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:
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
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
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The lock-and-key hypothesis
The induced-fit hypothesis
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The modified model of enzyme activity is known as the ‘induced-fit hypothesis’
Although it is very similar to the lock and key hypothesis, in this model the enzyme and substrate
interact with each other: Your notes
The enzyme and its active site (and sometimes the substrate) can change shape slightly as the
substrate molecule enters the enzyme
These changes in shape are known as conformational changes
This ensures an ideal binding arrangement between the enzyme and substrate is achieved
This maximises the ability of the enzyme to catalyse the reaction
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The induced-fit hypothesis
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Exam Tip
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Don't forget – our current understanding of enzyme-substrate interactions is based on the induced-fit
hypothesis.
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Measuring Enzyme Activity
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Measuring Enzyme Activity
The progress of enzyme-catalysed reactions can be investigated by:
Measuring the rate of formation of a product using catalase
Measuring the rate of disappearance of a 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:
Hydrogen peroxide is a common but toxic by-product of metabolism
This means it must be broken down quickly
Catalase is an enzyme found in the cells of most organisms that breaks hydrogen peroxide down
into water and oxygen
Hydrogen peroxide and catalase are combined and the volume of oxygen generated is measured
in a set time
The rate of reaction can then be calculated
Investigating Catalase Diagram
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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:
Amylase is a digestive enzyme that hydrolyses starch into maltose and glucose
Amylase functions best at pH 7 and 37oC (all enzymes operate best under specific optimal
conditions)
Amylase and starch are combined and this reaction mixture is then tested for starch at regular
time intervals
This can be done by taking samples from the reaction mixture at each time interval and adding
each sample to some iodine solution (starch forms a blue-black colour with this solution)
In this way, the time taken for starch to be broken down can be measured
The investigation can be repeated under a variety of conditions (e.g. by altering pH or
temperature) and the reaction rates can then be compared
Investigating Amylase Reaction
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Experimental set-up for investigating the rate of disappearance of a substrate using amylase
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Colorimetry
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Colorimetry to Measure Enzyme Activity
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
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:
1. 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)
2. 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)
3. 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
4. A calibration graph is then plotted of starch concentration (X-axis) vs percentage absorbance or
percentage transmission (Y-axis)
Serial Dilution Diagram
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Serial dilution of starch to make a range of concentrations
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