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Learning outcomes
• Understand the roles of enzymes as biocatalysts in living system
• Understand the factors affecting the activity of enzymes
Topic 4 - Enzyme
FSC BIO 124
Biology I
Chew Weiyun

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Enzyme Enzyme as Biological Catalyst

Enzymes
• Enzymes are protein molecules which can be defined as • Lowers activation energy
biological catalysts. • Speed up biochemical reaction rate
• Remains unchanged at the end of reaction
• A catalyst is a molecule which speeds up a chemical reaction • Globular proteins (tertiary structure of protein)
but remains unchanged at the end of the reaction.
• A metabolic pathway is a number of reactions
catalysed by a sequence of enzymes
• Many enzyme names end in -ase – for example amylase and
Know your terms?
ATPase.  Substrate
Tertiary structure of enzyme

 Products
 Enzyme-substrate
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Enzymes reduces activation energy Enzyme

• Firstly, reactants much reach a
high energy transition state • The overall process can be summarised as follows:
before changing into products
• Energy required to reach this
state is called activation energy
• Enzyme provides alternative pathway
• Enzyme binds to substrate forming
enzyme-substrate complex which
corresponds to transition state
(lowering the activation energy)

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Mechanism of Enzyme Lock & Key Hypothesis

• The shape of the active site allows the substrate to fit perfectly.
Enzymes • The idea that the enzyme has a particular shape into which the substrate fits
exactly.
• Enzymes are globular proteins. • Substrate (Key), enzyme (lock), enzyme-substrate complex
• Like all globular proteins, enzyme molecules are coiled into a precise
three dimensional shape, with hydrophilic R groups (side chains) on
the outside of the molecule ensuring that they are soluble.
• Has a groove called active site
• The active site of an enzyme is a region, usually a cleft or depression,
to which another molecule or molecules can bind.
• Active site and substrate are exactly complementary

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Lock & Key Hypothesis
1) An enzyme has a cleft in its
surface, called the active
site. The substrate
molecule has a
complementary shape.
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Lock & Key Hypothesis
3) The interaction of the
substrate with the active site
breaks the substrate apart. An
enzyme–product complex is
briefly formed, before the two
product molecules leave the
active site, leaving the enzyme
molecule unchanged and
ready to bind with another
substrate molecule.
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Lock & Key Hypothesis
2) Random movement of
enzyme and substrate brings
the substrate into the active
site. An enzyme–substrate
complex is temporarily
formed. The R groups of the
amino acids in the active site
interact with the substrate.
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Induced-fit Hypothesis
• Modified from lock & key
hypothesis
• Widely accepted
• adds the idea that the enzyme,
and sometimes the substrate,
can change shape slightly as
the substrate molecule enters
the enzyme, in order to ensure
a perfect fit.
• As a result, this makes the
catalysis even more efficient.
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Induced-fit Hypothesis Induced-fit Hypothesis

1. Binding of substrate causes slight change to the shape of the
enzyme, making the fit more precise
2. The active site now becomes fully complementary with the
substrate
3. The fit brings the molecules in close proximity and in correct
orientation for reaction to take place
4. Also causes stressing and distortion of chemical bonds (substrate) -
> lowers the activation energy

At the end of reaction, enzyme structure is unchanged and can be reused

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The course of an enzyme-catalysed reaction The course of an enzyme-catalysed reaction

1) When the enzyme and 2) More and more substrate is
substrate are first mixed converted into product
• there are a large number of • there are fewer and fewer
substrate molecules. substrate molecules to bind
with enzymes.
• Every enzyme molecule has a
substrate molecule in its • Enzyme molecules may be
‘waiting’ for substrate
active site. molecules to hit their active
Rate is dependent on:- sites.
• The speed enzyme converts • Fewer substrate, reaction gets
substrate to product, release slower.
it, and bind with another. • Until it stops.

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Factors that affect enzyme action Factors that affect enzyme action
The effect of Substrate concentration
The effect of enzyme concentration • [substrates] increases, here are greater
• [enzymes] is increased, more available chances of collision with enzyme.
active sites. • More enzyme-substrate complexes are
• More enzyme-substrate complexes are formed, more products are formed and the
formed, more products are formed and rate of reaction is increased.
the rate of reaction is increased.
• Then, limiting factor is the enzyme • The limiting factor is the substrate
concentration. concentration.
• Once all substrates have formed enzyme- • Once all enzymes are occupied and working
substrate complexes, a further increase in at maximum turnover rate, a further increase
concentration will have no effect on the in concentration will have no effect on the
rate of reaction. rate of reaction.
• Then, limiting factor is the substrate • At this point, the limiting factor is the enzyme
concentration. concentration.

Chew Weiyun *Initial volume of extract = enzyme concentration Chew Weiyun *Initial volume of extract = enzyme concentration

Factors that affect enzyme action Factors that affect enzyme action
The effect of Substrate concentration The effect of pH
• The enzyme is working at its maximum • Any change in the pH value of the
possible rate, known as Vmax. medium around the enzyme will cause
ionic and hydrogen bonds to be
• V stands for velocity. damaged, this will change the 3-D
shape of the enzyme and deform the
active site.
• The lower the pH, the higher the
hydrogen ion concentration. Hydrogen
ions can affecting hydrogen and ionic
bonding between R group of protein.
• Changes the shape of the active site.

Chew Weiyun *Initial volume of extract = enzyme concentration Chew Weiyun *Initial volume of extract = enzyme concentration
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Factors that affect enzyme action Factors that affect enzyme action
The effect of pH The effect of Temperature
• The substrate will therefore not be able
to fit into active site so the reaction
slows down or stops.
• The effects of pH is reversible within
certain limits but if the pH is far from
optimal value, the enzyme gets
denatured.

Chew Weiyun *Initial volume of extract = enzyme concentration Chew Weiyun *Initial volume of extract = enzyme concentration

Factors that affect enzyme action Factors that affect enzyme action
The effect of Temperature The effect of Temperature
• As the temperature increase, the kinetic • If the temperature continues to
energy and the enzyme activity increase increase beyond optimal
as well until optimal temperature is temperature, the rate of the reaction
reached (usually 40 degrees).
decrease as more kinetic energy
• Kinetic energy increases, enzyme and breaks the hydrogen bonds in the
substrate moves faster. Collisions secondary and tertiary structure of
happen more frequently. enzyme.
• High kinetic energy, bond easier to bond • At first, the substrate molecule fits
or broken. less well into the active site of the
• At optimal temperature, maximum rate enzyme, the rate of the reaction
of reaction is achieved. begins to slow down.

Chew Weiyun *Initial volume of extract = enzyme concentration Chew Weiyun *Initial volume of extract = enzyme concentration
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Factors that affect enzyme action Factors that affect enzyme action
The effect of Temperature
• Eventually, the substrate to no longer Some microbes (thermophile)
fit. have high optimal
• The enzyme is denatured. temperatures while cold area
microbes (psychrophiles) have
• Irreversible low optimal temperatures –
enabling them to survive in
their habitat

Chew Weiyun *Initial volume of extract = enzyme concentration Chew Weiyun

Enzyme inhibitors Enzyme inhibitors

• Binds to enzyme either to stop reaction or decrease activity (slow Competitive, reversible inhibition
down a reaction) • Inhibitor molecule have similar shape
• Inhibitors can be permanent (irreversible) or temporary (reversible) with natural substrate
• For this syllabus, we will focus on reversible inhibitors. • They fit in temporarily to compete
with the same enzyme active site
• 2 types: • Can be reversed by adding more
substrate
i. Competitive, reversible inhibitors
ii. Non-competitive, reversible inhibitors

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Enzyme inhibitors Enzyme inhibitors

Competitive, reversible inhibition Competitive, reversible inhibition
• [Substrate] much more than
[Inhibitor]
(a) Action of the enzyme • substrate molecules can easily bind to
succinate dehydrogenase on the active site in the usual way, and so
succinate the enzyme’s function is unaffected.
• [Inhibitor] increase or [substrate]
decrease
• it becomes less and less likely that the
(b) Competitive inhibition of substrate will collide with an empty
succinate dehydrogenase site. The enzyme’s function is then
inhibited.
by malonate
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Enzyme inhibitors Enzyme inhibitors

Competitive, reversible inhibition
Example
• IF a person accidentally drunk ethylene glycol.
• Ethylene glycol is used as antifreeze.
• Ethylene glycol is rapidly converted in the body to oxalic acid, which
can cause irreversible kidney damage.
• However, ethanol can act as a competitive inhibitors for the active
site of the enzyme that converts ethylene glycol.
Solution for ethylene glycol toxicity?
Non-competitive, reversible inhibitors
• Do not bind to the active site of enzyme; but
binds to allosteric site on the enzyme
• Allosteric site is located on the other site on
an enzyme (activators or inhibitors)
• The binding causes change in conformation of
enzyme molecule and its active site
• Disrupt the normal arrangement of hydrogen bonds
and hydrophobic interactions that holds the enzyme in
3D shape.
• Substrate is unable to bind with the active site
They are not competing for the active site

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Enzyme inhibitors Enzyme inhibitors

Non-competitive, reversible inhibitors
• Increasing substrate concentration does not
affect the rate of reaction

• E.g: cyanide attaches itself to the copper

prosthetic group of enzyme cytochrome
oxidase and inhibiting respiratory
reactions

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Enzyme inhibitors Enzyme inhibitors

Non-competitive, reversible inhibitors Non-competitive, reversible inhibitors
• An end product can act as an non-competitive, reversible enzyme inhibitiors • Example
• For the regulation of metabolic pathway • ATP is produced by cellular respiration. When the concentration of
ATP is high, it acts as allosteric inhibitor and inhibits biochemical
Example of a feedback mechanism reactions. When ATP concentration falls, ATP leaves the allosteric site
and cellular respiration is no longer inhibited.

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Classification of Enzymes Classification of Enzymes

6 major classes
iv. Isomerases – catalyses the rearrangement of atoms within a molecule converting one
i. Oxidoreductases – catalyse redox reaction by transfer of H, O or electrons from isomer to another
molecule to another phosphoglucoisomerase
Glucose oxidase Glucose-6-phosphate Fructose-6-phosphate
Glucose + Oxygen Gluconic acid + water

v. Lyases – breaking chemical bonds without addition of water

ii. Hydrolases – hydrolysis of substrate by addition of water
Pyruvate decarboxylase
Sucrase Pyruvate Ethanol + Carbon dioxide
Sucrose + Water Glucose + Fructose
vi. Ligases – catalyse reaction in which new chemical bonds are formed and uses ATP as
iii. Transferases – transfer of specific group of atoms from one molecule to another an energy source
ATP AMP + Pi + Pi
Hexokinase
Glucose + ATP glucose-6-phosphate + ADP Amino acid + tRNA Amino acid-tRNA complex
Aminoacyl-tRNA synthetase

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Enzyme kinetics Enzyme kinetics

• Turnover rate – number of substrate converted into product per • To understands how well an enzyme performs:-
seconds • Deduction from Michaelis-Menten constant (Km) and maximum velocity of the
reaction (Vmax)

• The enzyme carbonic anhydrase is one of the fastest enzymes known.

It can remove 600 000 molecules of carbon dioxide from respiring
tissue per second, roughly 107 times as fast as the reaction would
occur in the absence of the enzyme.

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Enzyme kinetics Enzyme kinetics

• This is done by plotting the velocity of
reaction against the substrate
concentration.
In the graph
• At Vmax all the enzyme molecules are
bound to substrate molecules – the
enzyme is saturated with substrate.
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Michaelis–Menten constant (Km )
• The Michaelis–Menten constant (Km)
is the substrate concentration at which
an enzyme works at half its maximum
rate (½Vmax).
• At this point, half the active sites of the
enzyme are occupied by substrate.
• The higher the affinity of the enzyme
(Km ↓) for the substrate, the lower the
substrate concentration needed for
this to happen.
Lower [substrate]
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Enzyme kinetics Enzyme kinetics

Michaelis–Menten constant (Km ) Michaelis–Menten constant (Km )
• The Michaelis–Menten constant (Km)
measures the affinity of the enzyme for 𝑉 [𝑆]
the substrate. 𝑉=
𝐾 + [𝑆]
• The higher the affinity (Km ↓) , the V = velocity of the reaction
more likely the product will be formed Vmax = maximum velocity
when a substrate molecule enters the [S] = substrate concentration
active site, rather than the substrate
simply leaving the active site again
before a reaction takes place.

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Enzyme kinetics Enzyme kinetics

Lineweaver-Burke plot The significance of Vmax and Km values
• Reciprocal of Michaelis-Menten formula. • Vmax - measures the maximum rate of reaction that is possible
• Km - measures the affinity of the enzyme for the substrate.
= × + (y=ax+c) • Vmax and Km values are independent of each other.
[ ]
• Plot the graph of (1/v) against (1/[S]) • Km is inversely proportional to affinity.
• Low Km has high affinity to substrates.
• Vmax – directly proportional to the enzyme concentration when other factors
are constant.
• Vmax reveals the maximum turn over number, the number of substrate
molecule that is converted to product per enzyme molecule per minute under
fixed set of conditions.

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Enzyme kinetics Enzyme kinetics

• The value of Km for a particular enzyme can vary, depending on a • Turnover numbers, which are related to Vmax and Km values
number of factors:-
• the identity of the substrate • overall ion concentration
• temperature • the presence of poisons
• pH • pollutants
• presence of particular ions • inhibitors.

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Enzyme kinetics
Competitive inhibitor
• With a competitive inhibitor, the
reaction can eventually reach its
normal Vmax, but it takes a higher
concentration of substrate to get
it there.
• In other words, Vmax is
unchanged, but the
apparent Km is higher. (affinity
lowered)
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Enzyme kinetics
Noncompetitive inhibitor
• With a noncompetitive inhibitor,
the reaction can never reach its
normal Vmax, regardless increase
in [substrate].
• A group of the enzyme
molecules always be inhibited,
so the effective concentration of
enzyme is reduced, Vmax is
reduced.
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Enzyme kinetics
Competitive inhibitor
Why must more substrate be
added in order to reach Vmax?
The extra substrate makes the
substrate molecules abundant
enough to consistently overcome
the effect of inhibitor to the
enzyme.
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Enzyme kinetics
Noncompetitive inhibitor
• However, the reaction reaches
half of its new Vmax at the same
substrate concentration, so Km is
unchanged.
• The unchanged Km reflects that
the inhibitor doesn't affect
binding of enzyme to substrate,
just lowers the concentration of
usable enzyme.
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Enzyme kinetics
The Lineweaver-Burk plots for inhibition

Thank you and all the best!!

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