Cambridge International AS & A Level Biology

3 Enzymes Key Terms

Globular proteins
3.1 Mode of action of enzymes
Active site
MISSESTRUCH 2024
Unique in shape

Enzymes are biological catalysts, made of globular Tertiary structure

proteins. They speed up reactions, without being
Intracellular
used up themselves. Some of the essential
reactions they catalyse include respiration, Extracellular
photosynthesis, protein synthesis and digestion.
Whilst enzymes are relatively large molecules, it is only a small part Catalase
of the enzyme that attaches to a substrate to catalyse a reaction. Trypsin
This site is known as the active site. The active site is specific and
unique in shape due to the specific folded and bonding in the tertiary structure of the protein. Due to
this specific shape active site, enzymes can only attach to substrates that are complementary in shape.

Enzymes catalyse intracellular and extracellular reactions. For example, catalase is an intracellular
enzyme inside liver cells that breaks down hydrogen peroxide into oxygen and water. Trypsin is an
extracellular enzyme, in the small intestines, that hydrolyses proteins.

Two Models of Enzyme Action

All reactions require a certain amount of energy before they occur,
this is known as the activation energy. When enzymes attach to
the substrate they can lower the activation energy needed for the
reaction to occur and therefore speed up the reaction.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.1 Mode of action of enzymes Enzyme

MISSESTRUCH 2024 Substrate

There are two hypotheses to explain how enzymes lower the Tertiary structure
activation energy.
Complementarity
shape
Lock and Key Model Hypothesis
Enzyme-substrate
complex
This model suggests that the enzyme is like a lock and that the
substrate is like a key that fits into it due to the enzyme’s specific Enzyme-product
tertiary structure resulting in a complementarity shape. This
complex
model suggests that the enzyme active site is a fixed shape and
that due to random collisions, the substrate can collide and attach to the enzyme. This forms an
enzyme-substrate complex and the charged groups within the active site are thought to distort the
substrate and therefore lower the activation energy. The substrate then reacts and the product is made,
creating an enzyme-product complex. The products are then released and the enzyme can be reused.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.1 Mode of action of enzymes Enzyme active site

MISSESTRUCH 2024 Induced

There are two hypotheses to explain how enzymes lower the Enzyme-substrate
complex
activation energy.
Enzyme moulding
Induced Fit Hypothesis Lowers the
activation energy
This hypothesis suggests that the enzyme is like a glove and the
Enzyme-product
substrate is like your hand; the empty glove is not exactly complex
complementary in shape to your hand, but when your hand enters
it enables the glove to mould around your hand to become
completely complementary. This analogy describes the process well. Induced fit is when the enzyme
active site is induced, or slightly changes shape, to mould around the substrate. When the enzyme-
substrate complex occurs, due to the enzyme moulding around the substrate it puts strain on the bonds
and therefore lowers the activation energy. The substrates react and the products form, creating the
enzyme-product complex. The products are then removed, and the enzyme active site returns to its
original shape.
The induced fit hypothesis is based on more recent research and is the currently accepted hypothesis.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.1 Mode of action of enzymes Products

MISSESTRUCH 2024 Measuring cylinder

Data logger
Investigating
Enzyme-Catalysed Reactions

Catalase Reaction Monitoring

Catalase is an enzyme that catalyses the breakdown of hydrogen peroxide

(substrate) into water and oxygen. To investigate the progress of this reaction,
we can measure the rate of formation of products, namely oxygen, over time.
This can be achieved by collecting the oxygen gas evolved during the reaction in
a calibrated measuring cylinder and recording the volume at regular time
intervals.
Alternatively, the rate of oxygen production can be indirectly measured by using a gas pressure sensor
connected to a data logger, which records changes in pressure as oxygen is produced.
By plotting the volume or pressure of oxygen produced against time, a graph can be generated to visualise
the rate of the catalase-catalysed reaction.

Amylase Reaction Monitoring

Amylase is an enzyme that catalyses the hydrolysis of starch (substrate) into simpler sugars such as
maltose and glucose.

To monitor the progress of this reaction, we can measure the rate of disappearance of the starch
substrate over time.

This can be accomplished by taking samples of the reaction mixture at regular intervals and testing for
the presence of starch using an iodine solution.
As the starch is broken down into simpler sugars, the iodine solution will change from blue-black to
orange/brown, indicating the depletion of starch.
By quantifying the intensity of the remaining colour or lack thereof using a colorimeter the rate of starch
disappearance can be determined.
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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.1 Mode of action of enzymes Kinetic energy

MISSESTRUCH 2024 Enzymes denature

Enzyme-complexes
Using a Colorimeter for Tertiary structure
alters
Measuring Enzyme-Catalysed
Shape
Reactions:
Principle of Colorimeter
A colorimeter is an instrument used to measure the absorbance or transmittance of light by a solution.
It works on the principle that certain substances absorb light at specific wavelengths, resulting in a
change in the intensity of transmitted light.
Enzyme-catalysed reactions often involve colour changes due to the formation of reaction products or
loss of substrates, which can be measured using a colorimeter.

Factors Affecting Enzymes

Enzymes are globular proteins sensitive to certain conditions. The following conditions affect the rate of
enzyme-controlled reactions:
1. Temperature
2. pH
3. Enzyme concentration
4. Substrate concentration

Temperature

If the temperature is too low there is insufficient kinetic energy

for successful collisions. If the temperature is too high enzymes
denature, the active site changes shape and enzyme-complexes
cannot form. Enzymes denature at high temperatures because
the increase in temperature causes bonds to break and the
tertiary structure alters, causing a change in the shape of the
active site.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.2 Factors that affect enzyme Tertiary structure
alters
action
Shape
MISSESTRUCH 2024
Denatures

pH Optimal pH

Fewer collisions
Too high or too low a pH will
interfere with the charges in the Rate of reaction
amino acids in the active site.
This causes bonds to break and
Plateau
the tertiary structure alters, Enzyme-substrate
causing a change in the shape of complexes
the active site.

Therefore, the enzyme denatures. Enzymes have different optimal pH values that they work at. For
example, pepsin has an optimum of around pH 2 and this is an advantage as it digests proteins in the
stomach which is acidic.

Substrate and Enzyme Concentration

If there is a low concentration of substrate, the reaction

will be lower as there will be fewer collisions between
the enzyme and substrate. Increasing the substrate
concentration will therefore increase the rate of
reaction. At high substrate concentrations, the rate of
reaction will plateau because all the enzyme active sites
are in use (the enzymes are saturated).

At low enzyme concentrations, there will be a lower rate of reaction. Increasing the enzyme
concentration will increase the rate of reaction as enzyme-substrate complexes will be more likely to
form. At high enzyme concentrations, unless unlimited substrate is added, the rate of reaction plateaus
as there will be insufficient substrate to bind with the large number of enzymes.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.2 Factors that affect enzyme Competitive
inhibitors
action
Same shape as
MISSESTRUCH 2024 the substrate

Bind to the active

site
Enzyme Inhibitors
Enzyme-inhibitor
complexes
Non-competitive
inhibitors

Allosteric site

Active site to
change shape

Competitive inhibitors are the same/similar in shape as the substrate. This means the competitive
inhibitor is complementary in shape to the active site and can bind to the active site. This prevents the
substrate from binding and enzyme-inhibitor complexes form instead of enzyme-substrate complexes
and the rate of reaction is lower. Most competitive inhibitors are reversible, meaning they can be
removed. If a high enough concentration of substrate is added, the substrate can knock out the inhibitor
and therefore the rate of reaction will increase.

Non-competitive inhibitors bind to the

enzyme away from the active site called
the allosteric site. This causes the
Rate of reaction

active site to change shape, and

therefore the substrate can no longer
bind, regardless of how much substrate
is added. Far fewer enzyme-substrate
complexes form and the rate of
reaction is much lower.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.2 Factors that affect enzyme Reversible
action Non-reversible
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Non-competitive

Allosteric site
Reversible and Non-Reversible
Irreversible
Enzyme inhibitors can be reversible or non-reversible. inhibitors
Reversible means the inhibitor can be removed, whereas non- Medicine
reversible means the inhibitor cannot be removed from the
enzyme. Cyanide is an example of a non-reversible non- reversible inhibitors
competitive inhibitor. The inhibitor binds to the allosteric site
controlling the
on cytochrome oxidase, a carrier molecule in the electron reaction
transport chain of respiration. It, therefore, prevents ATP
production and eventually leads to death. Not all irreversible Vmax
inhibitors are harmful, and in fact, some are used as medicines.
Proton pump inhibitors are medicines which bind irreversibly to
the enzyme involved in the secretions of hydrogen ions into the stomach (a cause of chronic indigestion).
Therefore, these proton pump inhibitors reduce the overproduction of acid in the stomach and prevent
ulcers from forming.

End-Product Inhibition
The products of some reactions are reversible inhibitors for the enzymes involved in controlling the
reaction. This enables the reactions to be controlled so that the reaction only occurs when the product
has been used up and therefore more product is needed. If there is a lot of product present, it will inhibit
the enzymes and cause the reaction to slow or stop. This prevents resources from being wasted.

Maximum Rate of Reaction (Vmax)

Vmax refers to the maximum rate at which an enzyme catalyses a reaction when the active sites of all
enzyme molecules are fully saturated with substrate. At Vmax, the enzyme is functioning at its maximum
capacity and further increases in substrate concentration do not result in a higher reaction rate.

Vmax is a key measure used to characterise enzyme kinetics and is determined experimentally by
plotting reaction rate against substrate concentration until a plateau is reached.
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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.2 Factors that affect enzyme Km
action Affinity
MISSESTRUCH 2024 Substrate affinity

Michaelis–Menten Constant (Km)

The Michaelis–Menten constant, Km, is a measure of the affinity of an enzyme for its substrate. It
represents the substrate concentration at which the reaction rate is equal to half of Vmax.
Km can be thought of as the substrate concentration required to achieve half-maximal velocity.
Enzymes with lower Km values have a higher affinity for their substrates, as they reach half-maximal
velocity at lower substrate concentrations.
Km is derived from the Michaelis–Menten equation, which describes the relationship between reaction
rate, substrate concentration, Vmax, and Km.

Comparison of Enzyme Affinity

Km values can be used to compare the affinity of different enzymes for their substrates.
Enzymes with lower Km values exhibit greater substrate affinity and are more efficient at converting
substrate into product, even at low substrate concentrations.
Conversely, enzymes with higher Km values have lower substrate affinity and require higher substrate
concentrations to achieve maximal velocity.

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 Cambridge International AS & A Level Biology

3 Enzymes Key Terms

3.2 Factors that affect enzyme Immobilized
enzyme
action
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Investigating Enzyme Activity in Different Forms

Enzyme Immobilised in Alginate vs. Free in Solution

Enzymes can be immobilised within a matrix such as alginate or

kept free in solution for various applications.
When investigating the difference in activity between these
forms, experiments can be conducted by comparing reaction
rates under controlled conditions.
For example, the activity of an immobilised enzyme can be
compared to that of the same enzyme in solution by measuring
the rate of substrate conversion to product over time.

Advantages of Using Immobilised Enzymes

More stable: Immobilised enzymes are often more stable than their soluble counterparts, as they are
protected from harsh environmental conditions and can be reused multiple times.
Ease of separation: Immobilised enzymes can be easily separated from the reaction mixture,
simplifying product purification.
Continuous use: Immobilised enzymes allow for continuous operation in bioreactors, as they can be
packed into columns or membranes for continuous flow systems.
Reusability: Immobilised enzymes can be reused for multiple
cycles, reducing the need for enzyme replenishment and lowering Progress Check
overall operational costs. Basic
Improved reactor performance: Immobilisation can improve
enzyme activity by providing a high local concentration of substrate Good
near the active site, leading to increased reaction efficiency. Ready

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3 Enzymes
References

https://commons.wikimedia.org/wiki/File:Catalyst_Energy_Diagram.png
https://commons.wikimedia.org/wiki/File:Two_substrates.png
https://commons.wikimedia.org/wiki/File:Induced_fit_diagram.svg
https://commons.wikimedia.org/wiki/File:Effect_of_temperature_on_enzymes.svg
https://commons.wikimedia.org/wiki/File:Enzyme-ph.png
https://commons.wikimedia.org/wiki/File:KinEnzymo(en).svg
https://commons.wikimedia.org/wiki/File:Competitive_inhibition.png
https://commons.wikimedia.org/wiki/File:Michaelis-Menten_Inhibitions.png
https://commons.wikimedia.org/wiki/File:Enzymes_immobilised_in_alginate_gel.jpg

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