AS Level
Chapter 3
Enzymes
Chapter Outline
Part 1: Enzyme Structure and Mode of Action
• Enzyme Structure
• Lock and Key Hypothesis vs Induced Fit hypothesis
• Mode of Action
Part 2: Mechanism of Enzymatic Reaction
• Measuring the rate of reactions
• Time course graph
Part 3: Factors Affecting Enzymatic Reaction
• Substrate concentration
– Maximum rate of reaction (Vmax)
– Michaelis-Menten constant (Km) – a measure of affinity
• Enzyme concentration
• Temperature
• pH
• Competitive vs Non-competitive Inhibition
Part 4: Immobilised enzymes Updated on 27/7/21 by Beh SJ @behlogy
Enzyme
Structure
What are enzymes?
• Biological catalysts
→ Can break down or
synthesize products
→ Used to control
metabolic reactions
Features:
• Speeds up / increase rate of chemical reaction
• Specific: 1 enzyme to only one / few substrates
• Unchanged at the end of the reaction
• Effective in small amounts
• High turnover number = can catalyse many reactions per unit time
Updated on 27/7/21 by Beh SJ @behlogy
How Do Enzymes Catalyze Reactions?
• Enzymes lower the activation energy of chemical reactions
• Activation energy (Ea) = Energy needed for a chemical reaction to
successfully form products
• This provides an alternative pathway for reactions to occur
Updated on 27/7/21 by Beh SJ @behlogy
All enzymes are globular proteins
• Spherical / ball shape
• Mostly tertiary structures, some quaternary
→ Have precise 3D structure due to interactions between R groups
• Soluble because…
→ Hydrophobic R groups are inside
→ Hydrophilic R groups faces outside
Primary structure → Tertiary structure
Updated on 27/7/21 by Beh SJ @behlogy
Active Site of Enzymes
All enzymes have an active site
• A site where substrates bind to
• Bind = complementary in shape
to substrate
• Active site gives specificity
• Only few catalytic amino acids
at the active site!
→ Precisely positioned
→ Not necessarily beside each
other in primary seq
Updated on 27/7/21 by Beh SJ @behlogy
How do substrates bind to enzymes?
More recent!
Lock and Key Mechanism Induced Fit Mechanism
Active site does not Active site is flexible and
change shape moulds around substrate
Shape of active site is Shape of active site is
fully complementary partially complementary
to shape of substrate to shape of substrate
Result: Substrate expected Result: Better fit!
to fit exactly!
Updated on 27/7/21 by Beh SJ @behlogy
Where do enzymes operate?
Some intracellular (inside cells)
• Cells synthesise enzymes and
retains them for internal use
Some extracellular (outside cells)
• Cells synthesise enzymes and
secrete them for external use
Updated on 27/7/21 by Beh SJ @behlogy
Mode of Action
Updated on 27/7/21 by Beh SJ @behlogy
Mode of Action
1. Enzymes and substrates move and collide randomly
• Only collisions in the right orientation
with enough energy result in a successful reaction
• E has active site with specific shape
that is complementary to substrate
Updated on 27/7/21 by Beh SJ @behlogy
Mode of Action
2. Formation of the enzyme-substrate complex (ESC)
• Substrates interacts with R groups of
catalytic amino acids at the active site
→ Forms temporary bonds
→ Active site changes shape to mould
around substrates (induced fit model)
→ Substrates binds strongly to active site
→ ESC is formed
Updated on 27/7/21 by Beh SJ @behlogy
Updated on 27/7/21 by Beh SJ @behlogy
Mode of Action
3. Product formation
• Interactions of substrates with the active site…
→ Brings substrates close together
in the right position
→ Put strain on the reactants
→ So bonds to break or form more easily
→ Allow transfer of charges/groups
→ Lowers activation energy
• Products form and leave active site
• Enzymes remain unchanged
Updated on 27/7/21 by Beh SJ @behlogy
Mechanism of Enzymatic Reaction
Updated on 27/7/21 by Beh SJ @behlogy
Rate of Reaction (ROR)
• Speed of conversion of substrate into product
• Rate is always per unit time
→ E.g. s-1 , min-1
• How to measure ROR in an experiment? By measuring the...
– Rate of product formation or
– Rate of substrate disappearance
• Steps:
1. Measure the product formation or substrate disappearance
2. Plot a time course graph
3. Find the gradient of the curve = ROR
Updated on 27/7/21 by Beh SJ @behlogy
Catalase
• Hydrogen peroxide is a toxic metabolic product in tissues
• Catalase enzymes are found in tissues of most living organisms
• We usually obtain it from potato
/ liver / yeast extract
• ROR = Rate of oxygen released
• Collect gas or count bubbles
Updated on 27/7/21 by Beh SJ @behlogy
Curve of Oxygen Produced Against Time
[P]
Volume of oxygen produced:
[P] increases then levels off
Speed of oxygen released:
Fast → slows down → stops
Rate of oxygen released:
High → decrease → 0
Rate = gradient
Gradient of a horizontal line = 0
Updated on 27/7/21 by Beh SJ @behlogy
Curve of Oxygen Produced Against Time
Initial rate of reaction
= gradient of the curve at 0s
Updated on 27/7/21 by Beh SJ @behlogy
Amylase
• Problem: Both substrate and products are colourless and are
both liquid!
• Answer: Use iodine test!
• ROR = Rate that starch disappears
Updated on 27/7/21 by Beh SJ @behlogy
Amylase
• Test samples with iodine solution at known time intervals
• Colour will change from dark blue → yellow/brown
Dark blue: Starch still present
Yellow/brown: Starch completely hydrolyzed
• Use spotting tile… or use a colorimeter to measure
the colour intensity
Updated on 27/7/21 by Beh SJ @behlogy
Amylase
• Use colorimeter measures light absorbance in arbitary units (a.u.)
• Measures relative absorbance to a control (distilled water)
Updated on 27/7/21 by Beh SJ @behlogy
Curve of Starch Remaining Against Time
[S] Concentration of starch, [S]:
Increases then levels off
Speed of starch being used:
Fast → slows down → stops
Rate of reaction:
High → decrease → 0
Rate = gradient
Gradient of a horizontal line = 0
Updated on 27/7/21 by Beh SJ @behlogy
Curve of Starch Remaining Against Time
Initial rate of reaction
= gradient of the curve at 0s
(or in this case, as early in
the reaction as possible)
Updated on 27/7/21 by Beh SJ @behlogy
What happens during
the course of the reaction?
To illustrate my next slide…
Pretty much the
same thing, right?
Updated on 27/7/21 by Beh SJ @behlogy
The Course of Reaction
Why was the rate high at first, then decreased and became 0?
High [S] → Low [S] → [S] = 0
S is in excess Lesser S available No S available for binding
Observe that:
• P is not shown here but [P] increases over time, then levels off
• [E] = constant
• [S] reduces over time, then levels off
• [S] the limiting factor in this reaction
Updated on 27/7/21 by Beh SJ @behlogy
Time Course Graphs
Rate is high bcs
more [S] is present
Note that [ES]
immediately
increases from
0 when the Rate is 0 bcs all S
reaction starts! has been used up
Updated on 27/7/21 by Beh SJ @behlogy
How to understand graphs?
• Always read the question carefully!
– Describe = state your observations
– Explain = explain why the observations are as such
– Compare / contrast = tell me the similarities or differences
between 2 sets of data
– CONTEXT matters!
• Read the axis labels!
– x-axis = independent variable
– y-axis = dependent variable
Updated on 27/7/21 by Beh SJ @behlogy
How to talk about graphs?
1. General trend
• First, describe how the independent variable and dependent
variable changes overall
E.g. when x increases, y increases then plateaus
• If needed, split the graph into several parts to describe and/or
explain separately
2. Comparative data quote
• Compare 2 points to support your statement
• Provide the x and y coordinates with the correct units
• You can also quote the max and min values where appropriate
• You can include some manipulative figures
E .g. the number of cases reduced by half in 2010 compared to 2008
Updated on 27/7/21 by Beh SJ @behlogy
Factors Affecting Enzymatic Reaction
Updated on 27/7/21 by Beh SJ @behlogy
Rate of Reaction
Rate of reaction be affected by:
1. Substrate concentration, [S]
2. Enzyme concentration, [E]
3. Temperature
4. pH
5. Inhibitors
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
What would happen if we use a higher [S]?
Higher [S]
Volume of oxygen
produced (cm3)
Lower [S]
How do we calculate the initial ROR?
We will find that the reaction is faster / ROR is higher for the
reaction with higher [S].
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Low [S] High [S]
[E] = constant
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
At low [S]
→ Some active sites available for binding / not all occupied
→ Few collisions between enzyme and substrate
→ Less S binds with active site
→ Few ESCs formed
→ [S] is limiting
• More active sites occupied as [S] increases
• Rate of reaction proportional to substrate concentration
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
At high [S]
• Rate increases to a plateau / Vmax reached
• Maximum rate of enzymatic reaction = Vmax
• All active sites are saturated
• Max no. of ESCs formed
• [E] is the limiting factor
• Further increase of [S] does not increase rate
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Low [S]
• Some active sites available
• [S] is limiting
Vmax
High [S]
• At Vmax, all active
sites are saturated
• [E] is limiting
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Initial Rate of
Reaction (cm3 s-1)
Vmax
Steps to find Km:
½ Vmax 1. Find half Vmax
2. Read [S] from x-axis
Km Substrate Concentration (mM)
(Michaelis-Menten
Constant)
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Michaelis-Menten Constant (Km)
• Km = [S] at ½ Vmax
• Km = the [S] at which the enzyme works at half its maximum rate
• At Km, ½ the active sites are occupied by S
• Km is a measure of enzyme affinity
• Affinity = the degree of attraction
between molecules
• The higher the Km, the lower the affinity
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Michaelis-Menten Constant (Km)
For an enzyme with high Km and low affinity:
• Higher [S] is needed to reach ½ Vmax
• Enzyme needs a higher [S] to reach Vmax
• Enzyme forms fewer ESC in the same unit of time
• Enzyme active site is a less good fit for substrate
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Michaelis-Menten Constant (Km)
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Vmax and Km
• Problem: too many experiments needed for the graph to
plateau in order to find Vmax and Km
• Solution: Plot a double-reciprocal graph!
→ Plot 1/v against 1/[S]
instead of v against [S]
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Double Reciprocal Plot
Updated on 27/7/21 by Beh SJ @behlogy
1) Substrate Concentration
Double-reciprocal plot
Updated on 27/7/21 by Beh SJ @behlogy
2) Enzyme Concentration
Low [E] High [E]
[S] = constant
Updated on 27/7/21 by Beh SJ @behlogy
2) Enzyme Concentration
At low [E], as [E] increases:
• More enzymes present
• More active sites available for S to bind
• Increase in frequency of collision
• More ESCs form
→ Rate increases as [E] increases
At high [E]:
• Rate of reaction levels off
• [S] is the limiting factor
• Max number of ESCs formed
Updated on 27/7/21 by Beh SJ @behlogy
2) Enzyme Concentration
Low [E]
• Less active sites available to bind to S
• [E] is limiting
High [E]
• Max number
of ESCs formed
• [S] is limiting
[S] = constant
Updated on 27/7/21 by Beh SJ @behlogy
3) Temperature
Optimum temp
Low temp High temp
[S] = constant
[E] = constant
Updated on 27/7/21 by Beh SJ @behlogy
3) Temperature
At low temp, as temp increases from low to
optimum:
• Increase in kinetic energy
• Enzyme and substrate molecules move faster
• Increase in collision rate between substrate
molecules and enzyme’s active site
• Substrates bind to enzyme’s active site more often
• More ESCs form
→ Rate increases as temp increases
Updated on 27/7/21 by Beh SJ @behlogy
3) Temperature
At optimum temperature:
• Maximum rate of enzyme reaction
• Most human enzymes ~40⁰C
• Different enzymes, different optimum temperature
E.g. enzymes in Thermophilic archaea have very high optimum temp
Updated on 27/7/21 by Beh SJ @behlogy
3) Temperature
At very high temp:
• Reaction slows down and then stops
• H bonds and ionic bonds
holding enzyme start to break
• Active site changes shape
• Enzyme is denatured
• Substrate cannot bind to the active site
• Fewer ESCs form
• Irreversible
Updated on 27/7/21 by Beh SJ @behlogy
3) Temperature
Low temp
• As temp increases,
kinetic energy increases
• More collisions Optimum temp
• More ESCs
High temp
• Enzyme denatures
• Ionic bonds and H
bonds break, active site
changes shape
[S] = constant
[E] = constant
Updated on 27/7/21 by Beh SJ @behlogy
4) pH
Optimum pH
Low pH High pH
[S] = constant
[E] = constant
Updated on 27/7/21 by Beh SJ @behlogy
4) pH
• pH = measure of the concentration of hydrogen ions in a solution
• Low pH – acidic (more [H+], less [OH-])
• High pH – alkaline (less [H+], more [OH-])
At low/high pH:
• H+ or OH- can interact with the R groups of
amino acids in enzymes
• Disrupt ionic bonds and H bonds
• Changes the active site shape
• S cannot bind to active site
• Less ESCs form
Updated on 27/7/21 by Beh SJ @behlogy
4) pH
At optimum pH:
• Max rate of enzyme reaction
• Beyond optimum pH - Enzyme denatures
• Different enzymes, different optimal pH
Updated on 27/7/21 by Beh SJ @behlogy
4) pH
Optimum pH
At Low/High pH:
• Ionic bonds and H bonds break
• Active site change shape
• Less ESCs form
[S] = constant
[E] = constant
Updated on 27/7/21 by Beh SJ @behlogy
5) Inhibitors
• Molecules which can reduce rate of an enzyme
catalysed reaction
Places where inhibitors can bind:-
1) Bind to enzyme’s active site
• Similar to substrate’s shape
• Competitive inhibitor
2) Bind to allosteric site of enzyme
• Another site on enzyme other than the active site
• Non-competitive inhibitor
Updated on 27/7/21 by Beh SJ @behlogy
5) Inhibitors
• Two types:
Updated on 27/7/21 by Beh SJ @behlogy
Competitive Inhibitors
• Fit into enzyme’s active site
• Similar shape to real substrate
• Competitive inhibitor competes with substrate for active site
of enzyme
• Reversible
Updated on 27/7/21 by Beh SJ @behlogy
Competitive Inhibitors
At low [S]:
• [inhibitor] > [substrate]
• Less frequency of collisions with S
• Less ESCs forms
• Reduces rate of reaction
→ Enzyme’s function inhibited
Updated on 27/7/21 by Beh SJ @behlogy
Competitive Inhibition
• Can be reversed/overcome by…. having more substrates
At high [S]
• [S] > [inhibitors]
• Inhibitor has less effect
• Substrate outcompetes inhibitor at high [S]
• More chances of substrate molecule
colliding and binding to active site
• More S binds to active site
• More ESCs form
→ Enzyme’s function unaffected
• Competitive, reversible inhibition
Updated on 27/7/21 by Beh SJ @behlogy
Competitive Inhibition
Vmax stays the
same bcs it is
determined by [E]
Km increases
when competitor is present
More [S] is needed to reach 1/2 Vmax
Updated on 27/7/21 by Beh SJ @behlogy
Non-competitive inhibition
Two types of non-competitive inhibition:
• Irreversible – e.g. in poisons (cyanide binds to enzyme in mitochondria
and inhibits ATP synthesis)
• Reversible – end product inhibition
Updated on 27/7/21 by Beh SJ @behlogy
Irreversible Non-Competitive Inhibition
• Inhibitor binds to allosteric site on enzyme
• Allosteric site = another site on enzyme
other than the active site
• Changes active site’s shape
• Disrupts H bonds and hydrophobic
interactions
→ 3D shape of enzyme affected
→ Distortion ripples
• Substrate unable to bind to active site
→ Enzyme’s function - inhibited
• Increasing substrate concentration has no
effects on enzyme activity
Updated on 27/7/21 by Beh SJ @behlogy
Irreversible Non-Competitive Inhibition
Vmax decreases
Inhibition is not reversible by increasing [S]
Lower [E] available for use
Km remains constant
Same [S] needed to reach Vmax
Updated on 27/7/21 by Beh SJ @behlogy
Graphs
No inhibitor
Vmax
Competitive inhibitor
(Same Vmax , Higher Km )
Vmax
½ Vmax Non-competitive inhibitor
(Lower Vmax , Same Km)
½ Vmax
Km Km
Updated on 27/7/21 by Beh SJ @behlogy
End Product Inhibition
• A form of control for
metabolic reactions
• Maintain homeostasis
In a chain of reactions
• Catalyzed by various
enzymes
• End product becomes a
non-competitive enzyme
inhibitor for upstream
reactions
Updated on 27/7/21 by Beh SJ @behlogy
End Product Inhibition
• High amount of end product
→ binds to allosteric site on another enzyme catalyzing a
reaction in the same metabolic chain
→ Inhibits reaction
• When amount of end product (inhibitor)
decreases, rate of reaction increases again
• Inhibitors can lose its attachment on
enzyme’s allosteric site and are used elsewhere
→ Temporary/brief
→ Non-competitive, reversible inhibition
Updated on 27/7/21 by Beh SJ @behlogy
Immobilised
Enzymes
Immobilised Enzymes
• Enzymes attached to an inert, insoluble material
How to encapsulate enzymes in alginate beads?
1. Mix enzymes with sodium alginate
2. Add drop by drop into calcium chloride
3. Pack them into a column
Updated on 27/7/21 by Beh SJ @behlogy
Immobilised Enzymes
4. Run substrates through the column
5. Collect products from bottom
You can run the products through the column again and again to
increase % yield.
Updated on 27/7/21 by Beh SJ @behlogy
Why Use Immobilised Enzymes?
• Can easily reuse enzymes
• Can easily recover enzymes
• Longer shelf-life of enzyme
• Less purification / downstream processing needed
→ Bcs it keeps product enzyme-free so no effect on product quality
• Allow continuous production
→ Can obtain more product per unit time
• Reduces end product inhibition
• Enzymes more tolerant of pH changes
• Thermostable – less likely to denature at high temp
Updated on 27/7/21 by Beh SJ @behlogy
Tolerance to pH and temperature
• Alginate protects enzyme
→ As enzyme is less exposed to solution
→ Less H+ or OH- penetrate the alginate beads
→ Shape of active site of immobilised enzyme is less disrupted
• 3D structure of enzyme is stabilized
→ H bonds vibrate less at high temp
→ Fewer bonds within immobilised
enzyme break
→ Active site less likely to change shape
→ Less denaturation
Updated on 27/7/21 by Beh SJ @behlogy
Chapter Outline
Part 1: Enzyme Structure and Mode of Action
• Enzyme Structure
• Lock and Key Hypothesis vs Induced Fit hypothesis
• Mode of Action
Part 2: Mechanism of Enzymatic Reaction
• Measuring the rate of reactions
• Time course graph
Part 3: Factors Affecting Enzymatic Reaction
• Substrate concentration
– Maximum rate of reaction (Vmax)
– Michaelis-Menten constant (Km) – a measure of affinity
• Enzyme concentration
• Temperature
• pH
• Competitive vs Non-competitive Inhibition
Part 4: Immobilised enzymes Updated on 27/7/21 by Beh SJ @behlogy
Useful Videos
• Explaining enzyme inhibitors and the graphs
[Link]
Links below cover things you SHOULD NOT write in exams
but maybe helpful for your understanding / for your entertainment!
• How to speed up chemical reactions (and get a date)
[Link]
• How do you explain Michaelis Menten to a kid?
(this is more detailed than needed)
[Link]
Updated on 27/7/21 by Beh SJ @behlogy