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CHAPTER 3 1
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 Types Of Enzymes
• exist throughout our entire body system - in
our organs, bones, blood and cells.
Metabolic • Grow new cells and maintain every tissue in
our body. When these enzymes are healthy,
enzymes robust and are present in adequate numbers,
they will do their job well

• are secreted by our various body organs - by

our salivary glands, stomach, pancreas and
Digestive small intestine.
• Help in the digestion of our food.
enzymes
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 Types Of Enzymes
METABOLISM

Catabolism Anabolism
(break down) (synthesis)

• Break down into simpler • Synthesis of complex

molecules with release molecules which
of energy requires energy.
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 Types Of Enzymes
• Intracellular enzyme
• Produced inside cells
• Catalyze reactions within cells

• Extracellular enzyme
• Secreted by cells
• Catalyze reaction outside cells

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 What are enzymes??
• Protein molecule
• Globular protein - Coiled 3D, spherical
Solubility - Hydrophilic R group of the
amino acids face outwards
of the protein molecule
- Hydrophobic R group…face
inwards …
• Active site – Hydrophobic depression for
Specificity substrate molecule(s) to bind
specifically, form bonds with R
group of amino acids, catalyse
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 Characteristics
• Biological catalyst
– Speeds up a chemical reaction by lowering
the activation energy
• Remains unchanged at the end
• Required in small amount
• Specific – active site

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 Induced-fit
hypothesis

How
enzymes Mechanism
work?? of enzyme
action

Lock-and-key
hypothesis

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 Lock-and-key
hypothesis
• Enzyme specific in reaction – active site
• Shape of substrate fits specifically (complementary) to
active site.
• Substrate binds with active site - hydrogen bonding
(temporary)
• Interaction of R groups form bonds with substrate
• E-S complex forms
• causes strain in substrate
• lowers activation energy
• breaks substrate bond, form new bonds to form products
**Greatly affected by competitive inhibitors
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• In practice, enzyme unlike rigid lock Induced-fit
hypothesis
• Enzymes are more flexible
• Substrate partially complementary to active site
• Enzyme molecules
• Slightly changed when binds with the substrates,
moulds to fit perfectly to the shape of substrate
• Stronger binding of substrate to active site
• Interaction of R groups form bonds with substrate
• Forms E-S complex
• Enzyme puts strain on substrate molecule
• Thereby lower activation energy
• Breaks substrate bond, form new bonds to form products
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 Enzymatic Reaction
• The close fit brings the molecules close
together and in the correct orientation for
reaction to take place.
• It causes stressing and distortion of chemical
bonds of the substrates. This causes the bonds
to break and new bonds to form.
• This makes it easier for the substrate to be
changed into the product and hence, lowering
the activation energy needed.
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 Specificity
• Substrate specific • Group specific
• Conformation of active site, as • One enzyme can catalyze
well as properties and reactions for a variety of
positions of chemical groups in substrates with similar
R groups, ensures that only structural or chemical
complementary substrates will properties
enter active site • Explained by induced-fit
• Explained by lock and key hypothesis
hypothesis

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S2013/V22/Q4(d)(i)

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S2010/V21/Q1(c)

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W2014/V23/Q4

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Activation energy & Transition State

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Reactant must reach a high-
energy intermediate state calle
the transition state before th
products are formed.

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Enzymes reduce activation energy

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Energy release

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Energy absorbed

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 Enzyme
Inhibitor
concentration

Factors that
affect the rate
of reaction
Substrate
pH concentration

Temperature

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 • Increasing temperature will
increase the rate of reaction up Temperature
to an optimum rate
(highest reaction rate).

• Increasing temperature
increases E’s and S’s
kinetic energy.
• E and S collide more
often per quantity of
time.
• Form more E-S complex
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Rate at which reaction
decreases
- Due to denaturation
of enzyme molecules
- Increasing
temperature causes
Rate at which reaction
atoms to vibrate,
increases
causing bonds to
- Due to increased
break, changing the
kinetic energy of
shape of active sites
substrate and enzyme
molecules

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 Temperature

• Temperatures above the optimum cause

increasing enzyme molecule vibration, breaking
down internal bonds and destroying the active
site.
• The enzymes become denatured (lose its shape
and activity). 26
Enzymes are sensitive to pH.
• pH is a measure of hydrogen ion concentration
• Arrangement of active site is partly fixed by hydrogen and
ionic bonds between –NH2 and –COOH that make up an
enzyme
• The lower the pH..
– The higher the hydrogen ion concentration
– Hydrogen ion interact with R groups of amino acids
– affects ionic bonding between the groups
– Affects the 3D arrangement of enzyme molecule
– changes shape of active site
• Operates in a narrow range on pH
• Very extreme pH will denature enzyme pH
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• Increasing enzyme concentration increases the
number of active sites available to catalyze the
reaction, thus increases the rate of the reaction
(if substrate is in EXCESS or SUFFICIENT).

Enzyme
concentration 28
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 Substrate
concentration

• Initially, sharp increase in rate because there a plenty of

substrate and free active sites
• Then, rate slows down because of the limiting factor which is
enzyme concentration
• Later, rate remained constant (plateau) even with increasing
substrate concentration because all enzymes are now fully
saturated. Limiting factor is enzyme concentration.
• Vmax is reached.
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Measuring enzyme-catalyzed reactions:
 Rates of formation of products (for example catalase)
 High substrate, high enzyme
 A lot of substrate in contact with active sites on enzymes
 Substrates rapidly broken down, products increases
rapidly
 Eventually all active sites occupied
 Less and less substrate 31
32 B
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D
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How to determine the reaction rate of an enzyme?
Time course of an enzyme-catalysed reaction by
measuring:
 Rates of formation of products
 Rates of disappearance of substrate

examples..

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Measuring enzyme-catalyzed reactions:
 Rates of disappearance of substrate (for example using
amylase).
 High substrate, high enzyme
 A lot of substrate in contact with active sites on enzymes
 Substrates rapidly broken down, products increases rapidly
 Eventually all active sites occupied
 Less and less substrate 36
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 The course of a reaction
• The rate of enzymatic reaction is always fastest at
the beginning
• “Initial rate of reaction”
• Enzyme/ substrate – still available, not limiting

• How to calculate? Measure..

• Slope of tangent to the curve, as close to
time 0 as possible
• Amount of product in the first 30seconds
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The rate of reaction in the first 30 s for each enzyme concentration.

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 Enzyme inhibition
Enzyme inhibitor…
• Substances that directly or indirectly interfere with the
functioning of active site of an enzyme and reduces its
activity

• Binds to the active site COMPETITIVE

• Bind to other sites than active site NON-COMPETITIVE

• Binds permanently Non-Reversible inhibitors

• Binds temporarily Reversible inhibitors
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Competitive inhibition

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Non-competitive inhibition

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 Competitive Reversible
• Competitive inhibitors with similar shape as
substrate bind reversibly and temporarily to the
active sites, preventing the binding of substrate.

• Substrate and inhibitor compete for the active

site on enzyme.

• Addition of substrate can reverse inhibition and

increase binding of substrate to active sites
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 https://www2.fmovies.se/film/house-md-2.kk49/m5lr2z
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 Example of competitive inhibitors
• Ethylene glycol (antifreeze in automobiles, printer
toner) is broken down in the body into oxalic acid
(deadly poison) by the enzyme, alcohol dehydrogenase.
• First affect the central nervous system, then the heart,
and finally the kidneys. Ingestion of sufficient amounts
can be fatal if untreated
Ethylene glycol oxalic acid
alcohol dehydrogenase

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Ethylene Glycol Poisoning
• Alcohol (ethanol) acts as a
competitive inhibitor for alcohol
dehydrogenase.
• Giving the patient large amounts of
alcohol will cause the ethanol to
compete with ethylene glycol for
the active site of alcohol
dehydrogenase.
• Alcohol is the preferred substrate for
alcohol dehydrogenase so when it is
present, it binds with the enzyme.
After a while, the ethylene glycol is
harmlessly excreted in urine
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Example of competitive inhibitors
Sulfonamide
• Sulfonamide is an antibacterial agent
• WWII – fight microbial infection
• Sulfonamide similar in structure to PABA Para
amino-bezoic acid)
(Substance essential for bacteria to synthesize
folic acid for its growth)
• Sulfonamide act as competitive inhibitors to PABA
• DHPS(dihydropteroate synthase)
PABA Folic acid (Used by bacteria
(Substrate) DHPS (Product) for cell growth)
(Enzyme) 49
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 Example of competitive inhibitors -
Malonate
• Malonate poison inhibits enzyme succinate dehydrogenase
• Compete with substrate succinate which is important in
respiration

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 Non-competitive Reversible
• Non- competitive reversible inhibitors bind
reversibly and temporarily to the other sites of
the enzyme molecule
• Disrupting the normal arrangement of hydrogen
bonds and hydrophobic interactions holding the
enzyme molecule in 3D shape
• This distortion affects the active site
• Active site unable to bind to substrate
• While inhibitor is still attached, addition of
substrate CANNOT reverse inhibition
• Example – End-product Inhibition
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 End product inhibitor
- Negative feedback inhibition
• Enzymes with other sites (allosteric
site) on enzyme molecule are often
found at start of biosynthetic
pathways
• Enzyme is controlled by the final
product of the pathway
• Final product binds to the another
part of enzyme (allosteric site)
• Prevent substrate from binding
• End-product can loose attachment
to active site
• Allowing enzyme to reform active
state 53
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Graph showing the comparative effects of the
different inhibitors on substrate concentration

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• When a competitive inhibitor is present, the
enzyme activity is decreased compared to
when no inhibitor is present. As the substrate
concentration is increased, the activity
eventually becomes the same.
• You will notice that the optimum substrate
concentration is much higher.
• With a non-competitive inhibitor, the
inhibition is not dependent on the substrate
concentration and the effect is similar to an
irreversible inhibitor.
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 ON ACTIVE SITES OTHER SITES

TEMPORARY COMPETITIVE NON-COMPETITIVE

REVERSIBLE REVERSIBLE
ETHYLENE GLYCOL END PRODUCT INHIBITION
MALONATE

PERMANENT NON-COMPETITIVE NON-COMPETITIVE

IRREVERSIBLE IRREVERSIBLE
PENICILLIN
HEAVY METALS IONS
DFP
PARATHION
DIGITALIS

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 Mock AS 20017
But…

Similar shape,
going for active site
QUESTION:
Using the information in Fig 2.4, suggest a reason how
exemestane acts as a non-competitive inhibitor of aromatase.
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 Answer

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 Vmax and Km values
Significance of Vmax and Km values
• Enable computerized models of biochemical pathways/
behaviour of whole cell
• Quantitatively compare enzyme’s preference for
different substrate
• Understand what affects enzyme efficiency
• Compare the performance of the same enzyme from
different organisms
• Apply calculations to other fields of biochemistry such
as antibody-antigen binding
• Enable calculation of proportion of active sites occupied
by substrate.. By knowing Km 60
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 Comparing enzyme affinities
• Turnover rate
– Rate/ speed at which enzyme converts
substrate to product
• Vmax
– Theoretical maximum rate (velocity)
– At Vmax, all enzyme molecules are bound
to substrate (fully saturated)
– How to measure Vmax
– However… this curves never flattens
completely in practise. Theoretically it
flattens at infinite substrate
– So..we use “Double reciprocal plot” 61
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Remember..
Vmax

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Answer:

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 If i/Vmax is 20

• From graph:
- At 1/Vmax, 1/[S] is zero
- Therefore [S] is infinite
• If 1/Vmax is 20…
• The Vmax is 1/20 = 0.05
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• Another value can be obtained from graph:
• Michaelis-Menten constant
• Km

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Michaelis-Menten constant

• Km
1) Km is the substrate concentration at which enzyme
works at half its maximum rate (1/2 Vmax)
2) Half the active site of enzymes are occupied by substrate
3) Km is a measure of the affinity of the enzyme for its
substrates

• The higher the affinity,

The lower the substrate concentration needed for reaction,
The lower the Km
The quicker the reaction reaches maximum rate

• BUT! Vmax is not affected by Km

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S2016/V23/Q2

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Which of the four enzymes in Table 3.2 has the highest
affinity for its substrate?
Explain your answer.

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Z
Y
X

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 Same Vmax

More [S] left due to

presence of competitive
Less [S] left because no inhibitor
inhibitor, so more substrate
bound to active sites
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http://slideplayer.com/slide/7849630/

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 Enzyme
Immobilization
• There is a vast variety
Enzyme immobilized in a
of enzymes to
matrix of calcium alginate
catalyse industrial
beads
important chemical
reactions
• It is also much more
efficient and specific
• Enzymes can be
redesign through
protein engineering
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 Enzyme Immobilisation
• Immobilisation process
• Example 1:
• Calcium alginate beads with immobilized amylase
– Amylase (enzyme) is mixed with a solution of sodium
alginate
– This mixture is dripped (usually through a syringe) into a
solution of calcium chloride
– The sodium ions are displaced by the calcium ions,
resulting in the formation of hard, insoluble beads of
calcium alginate
– The alginate beads are left to harden further, then rinsed
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 Enzyme Immobilisation
• Using the immobilised enzymes:

amylase
enzymes
Starch
(substrate)

A column of beads Calcium alginate

beads coated with
immobilzed
maltose amylase
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 Enzyme Immobilisation
• Immobilisation process
• Example 2:
• Calcium alginate beads with immobilized lactase
– Lactase (enzyme) is mixed with a solution of sodium
alginate
– This mixture is dripped (usually through a syringe) into a
solution of calcium chloride
– The sodium ions are displaced by the calcium ions,
resulting in the formation of hard, insoluble beads of
calcium alginate
– The alginate beads are left to harden further, then rinsed
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 Enzyme Immobilisation
Immobilizing enzymes • Using the immobilized
enzymes

Enzyme +
sodium alginate

Calcium chloride

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 Advantages of using immobilized enzymes

– Enzyme can be recovered after use

– Enzyme does not contaminate product
– Enhanced stability of the enzyme molecule
–Immobilized enzyme more tolerant to:
• Temperature change
• pH change
–Because embedded, not easily denatured
– Substrate can be easily passed through the enzyme
several times
– Can immobilize more than one enzyme
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 Enzyme Immobilisation

• The use of dipsticks in the

quantitative measurement of glucose:
– Glucose is not detectable in the
urine of healthy mammals
– If blood glucose concentration
increases above “renal threshold”
»Glucose will not be fully
reabsorbed from filtrate
»Diabetics have variable
amounts of glucose in
their blood and urine
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