Revision on enzymes

On 28/9/2022

Dr.Nihal GABR
 Definition

Globular protein , acting as a biological cataylst

Which speed up chemical reaction , by lowering the activation energy
With specific shape of active site
Tertiary structure ( 3D ) to enable protein molecules to carry its metabolic function
being a biological cataylst . O

What makes enzymes specific in their function :

1. Specific sequence of amino acids in the primary structure

2. Determine the arrangement and the type of R groups

3. Which determine the types of bonds and interaction between R groups

4. Thus in turn, it determines the over all folding and coiling of the poly peptide …giving a
precise 3D shape ( tertiary structure which brings the amino acids close together )

5. So enzyme have specific shape of active site .

Explain lock and key mechanism

1. Enzymes have specific shape of active site complementary to to a substrate

2. So that the substrate fit in the active site and bind by temporary hydrogen bonds
3. Forming enzyme substrate complex causing a a strain / stress on substrate
4. So lowering the activation energy
5. The molecule is broken / got two molecules close together for the formation of
new bond
6. Product will no longer fit in the active site so being released .
Explain induced fit mechanism
1. The substrate is partially complementary to the active site .
2. Active site CHANGE ITS SHAPE ( CONFORMATIONAL CHANGE) slightly
when the substrate binds to it where it moulds and folds around the substrate.
3. Where the R groups of amino acids in the active site interact with substrate
…..strong binding of substrate to active site …….( stress and lowers the activation
energy )
4. So the active site and the substrate are now said to be complementary to each
other allowing BETTER FIT .
5. Forming ESC ….where the activation energy is lowered
6. This interaction can cause the break of substrate apart or allow the formation
of new bond between molecules ..forming a new one or more products leaving the
active site of enzyme unchanged

How enzymes lower the activation energy to speed up a chemical reaction ?

1. By providing an alternative pathway

2. By bring reactants close to the active
site
3. Where R groups of amino acids in
active site interact with substrate
4. Putting a stress and strain on
reactants so making it easier to break or
form a new bond .
Investigating the progress of enzyme catalysed reaction

1. Get a known concentration of enzyme and substrate

2. Set the experiment at constant temperature and pH
3. Take a sample every 2 mins
4. Plot a graph ….time in seconds / minutes on x axis and
( dependent variable) on y axis
5. Dependent can be increase in product concentration or decrease in
substrate concentration.
 Describe and explain the shape of the curve for an enzyme catalyzed reaction:

Describe :
1. Overall increase in the product concentration by time
2. Initial steep increase , followed by slower increase
then leveling off at ………

Measure initial rate

Initial rate is measured by volume / time during the first
30S / 1 min)

Explain :

Initial steep increase

. High substrate concentration and many free active-sites
so more collisions and more ESCs……not limiting ( substrate or enzyme) .

Slow increase
Decrease in substrate concentration where it turned into product
occupied active sites so less successful collision so less ESCs.

Leveling off
Substrate has been used up.

Factors affecting enzyme activity

1.substrate concentration
Describe

1.As the substrate concentration increase, rate of

reaction increase reaching to maximum at ……..
2.Where there is initial steep increase
3.Followed by a gradual increase till…..
4.The level off at ………
Explain

At low substrate concentration

Substrate concentration is limiting factor
Few collision between enzyme and substrate
More free active sites ( enzyme in excess )
So few ESCs

By increasing substrate concentration:

More collision …more active site s occupied / saturated

At higher subtsrate concentration

Enzyme concentration becomes a limiting factor
All active sites are occupied …maximum number of ESCs

2.Enzyme concentration
Describe
All following Explain why comparing initial rate of
same trend of reaction ……….as at the beginning of
initial steep
the reaction the substrate concentration
increase
is tthe same / constant , so not a
followed b y
aslower limiting factor …..as later on as the time
increase then pass, the substrate concentration will
level off vary
So by this way we can have fair test
Describe
and valid comparison

Initial rate of
reaction
Explain
increase with an More enzymes …so more active sites
increase i …so more successful collisions so
enzyme more ESCs.
concentration
 3.Temperature
Explain the shape of the graph by increasing
temperature above optimum :

1. Steep decrease in rate of reaction , as molecules

vibrate energetically to the extent that some bonds
holding the enzyme molecule in its precise 3D shape
starts to break
2. Mainly its the hydrogen bonds ( and ionic bond )
3.so the enzyme will lose the tertiary structure
4. The shape of active site will be lost due to change in
3D shape
5, the enzyme is irreversibly denatured .

4.pH
Describe

At pH 7 , its the optimum pH for the enzyme , where the

enzyme work the fasters
Above or below the optimum , there is a steep decrease in
rate of reaction .

Explain 1. Change in pH means a change in hydrogen ion concentration in a a solution

2. The charge on R groups of amino acids at active site will be changed
Ionisation of R groups
3. So hydrogen and ionic bonds which are needed / important in maintaining the tertiary structure of
enzyme , are broken .
So active site shape will be changed With the change in 3D shape
Enzyme will be denatured.
So substrate can no longer fit so no ESCs formed .
 Maximum rate ofenzyme catalysed reaction.
V max At V max all enzyme molecules are bound to substrate …all active
More ESCs
sites are saturated /occupied . Rate of Rx

Depends of enzyme concentration Fewer ESCs

Substrate concentration
Higher V max …..higher rate of reaction …..more collision ….more ESCs
The one with lower Vmax needs more substrate to reach the V max and occupy
all active sites

Frequency of collision is lower

Km Michaelias Menten constant ( km inversely proportional to affinity )

Km = Concentration of substrate at which we reach 1/2 Vmax

Measure the affinity of the enzyme to substrate

Where the higher the affinity of the enzyme to the substrate, the lower the substrate
concentration needed to reach the V max .
I.e it takes a lower substrate concentration to saturate the active sites ( free active
sites are more likely to be saturated )
Make it easier for the substrate to enter the active site .

By making the shape of active site more complemnatry

5. Enzyme inhibitors :
A) competitive inhibitor

1. Inhibitor has similar structure to the substrate , which is

complementary to the active site .
. The inhibitor compete with substrate on the active site
3. So the inhibitor bind to the active site , substrate can’t bind to
active site .
4. So the frequency of successful collision decrease, so fewer
ESCs
5. So at lower substrate concentration , so slower rate of
reaction .
6. So higher substrate concentration is needed to reach the V
max .
Effect on V max and Km

V max ..remain the unchanged ….where the inhibitor compete in

the active site , so reducing frequency of successful collisions , so
fewer ESCc .. thus upon increasing substrate concentration , the
inhibitor will have least effect , so V max remains the same . ..

Km ….value of km will, increase …..as there is a decrease in the affinity of the enzyme to
the substrate , as the competitive inhibitor compete on the active site with substrate , so
higher substrate concentration is needed to reach V max .

B) non competitive inhibitor

1. Inhibitor binds to a site other than the active site /allosteric site .
2. This will disrupt the arrangement of hydrogen bonds and
hydrophobic interactions holding the enzyme in a specific 3D shape
…….causing a change in the tertiary structure …change in shape if
active site
3. Substrate is unable to bind to the active site to form ECS…so
fewer ECSc
4. V max …is not reached even by increasing substrate
concentration.
But km remains the same . .
 Definition

Globular protein , acting as a biological cataylst

Which speed up chemical reaction , by lowering the activation energy
With specific shape of active site
Tertiary structure ( 3D ) to enable protein molecules to carry its metabolic function
being a biological cataylst .

What makes enzymes specific in their function :

1. Specific sequence of amino acids in the primary structure
2. Determine the arrangement and the type of R groups

3. Which determine the types of bonds and interaction between R groups

4. Thus in turn, it determines the over all folding and coiling of the poly peptide …giving a
precise 3D shape ( tertiary structure which brings the amino acids close together )

5. So enzyme have specific shape of active site .

Explain lock and key mechanism

1. Enzymes have specific shape of active site complementary to to a substrate

2. So that the substrate fit in the active site and bind by temporary hydrogen bonds
3. Forming enzyme substrate complex causing a a strain / stress on substrate
4. So lowering the activation energy
5. The molecule is broken / got two molecules close together for the formation of
new bond
6. Product will no longer fit in the active site so being released .
 Explain induced fit mechanism

1. The substrate is partially complementary to the active site .

2. Active site CHANGE ITS SHAPE ( CONFORMATIONAL CHANGE) slightly
when the substrate binds to it where it moulds and folds around the substrate.
3. Where the R groups of amino acids in the active site interact with substrate
…..strong binding of substrate to active site …….( stress and lowers the activation
energy )
4. So the active site and the substrate are now said to be complementary to each
other allowing BETTER FIT .
5. Forming ESC ….where the activation energy is lowered
6. This interaction can cause the break of substrate apart or allow the formation
of new bond between molecules ..forming a new one or more products leaving the
active site of enzyme unchanged

How enzymes lower the activation energy to speed up a chemical reaction ?

1. By providing an alternative pathway

2. By bring reactants close to the active
site
3. Where R groups of amino acids in
active site interact with substrate
4. Putting a stress and strain on
reactants so making it easier to break or
form a new bond .
Investigating the progress of enzyme catalysed reaction

1. Get a known concentration of enzyme and substrate

2. Set the experiment at constant temperature and pH
3. Take a sample every 2 mins
4. Plot a graph ….time in seconds / minutes on x axis and
( dependent variable) on y axis
5. Dependent can be increase in product concentration or decrease in
substrate concentration.
 Describe and explain the shape of the curve for an enzyme catalyzed reaction:

Describe :
1. Overall increase in the product concentration by time
2. Initial steep increase , followed by slower increase then
leveling off at ………

Enzyme concentration and

substrate concentrations not
Measure initial rate
Initial rate is measured by volume / time during
-
limiting factors

the first 30S / 1 min)

Explain :

Initial steep increase

. High substrate concentration and many free active-sites
so more collisions and more ESCs……not limiting ( substrate or enzyme) .

Slow increase
Decrease in substrate concentration where it turned into product
occupied active sites so less successful collision so less ESCs.

Leveling off
Substrate has been used up.

Factors affecting enzyme activity

1.substrate concentration
Describe
1.As the substrate concentration increase, rate of
reaction increase reaching to maximum at ……..
2.Where there is initial steep increase
3.Followed by a gradual increase till…..
4.The level off at ………
 Explain

At low substrate concentration

Substrate concentration is limiting factor
Few collision between enzyme and substrate
More free active sites ( enzyme in excess )
So few ESCs

By increasing substrate concentration:

More collision …more active site s occupied / saturated

At higher subtsrate concentration

Enzyme concentration becomes a limiting factor
All active sites are occupied …maximum number of ESCs

2.Enzyme concentration
Describe
Explain why comparing initial rate of
All following reaction ……….as at the beginning of
same trend of
the reaction the substrate concentration
initial steep
increase
is tthe same / constant , so not a
followed b y limiting factor …..as later on as the time
aslower pass, the substrate concentration will
increase then vary
level off
So by this way we can have fair test

Describe
and valid comparison

Initial rate of
reaction
increase with an
Explain
increase i More enzymes …so more active sites
enzyme …so more successful collisions so
concentration more ESCs.
3.Temperature
Explain the shape of the graph by increasing
temperature above optimum :
1. Steep decrease in rate of reaction , as molecules vibrate
energetically to the extent that some bonds holding the enzyme
molecule in its precise 3D shape starts to break
2. Mainly its the hydrogen bonds ( and ionic bond )
3.so the enzyme will lose the tertiary structure
4. The shape of active site will be lost due to change in 3D shape
5, the enzyme is irreversibly denatured .

4.pH
Describe

At pH 7 , its the optimum pH for the enzyme , where the

enzyme work the fasters
Above or below the optimum , there is a steep decrease in

&.
rate of reaction .
1. Change in pH means a change in hydrogen ion concentration in a a solution
-
i'
E Hi+ +

Explain R
H
+
2. The charge on R groups of amino acids at active site will be changed
=

Ionisation of R groups
3. So hydrogen and ionic bonds which are needed / important in maintaining the tertiary structure of
enzyme , are broken .
So active site shape will be changed With the change in 3D shape
Enzyme will be denatured.
So substrate can no longer fit so no ESCs formed .
 Maximum rate ofenzyme catalysed reaction.
V max At V max all enzyme molecules are bound to substrate …all active
More ESCs
sites are saturated /occupied . Rate of
Vmax reflects how fast
the enzyme can
catalyze the reaction
Depends of enzyme concentration reaction
in ax
Fewer ESCs

Substrate concentration

Higher vmax …..higher rate of reaction ……more collision …….more ESCs

The one with lower V max needs more substrate to reach V max

Km
Michaelias Menten constant ( km inversely proportional to affinity )

Km = Concentration of substrate at which we reach 1/2 Vmax

Measure the affinity of the enzyme to substrate.

Where the higher the affinity of the enzyme to the substrate , the lower the
concentration of substrate needed to reach the V max
I.e it takes a lower substrate concentration to saturate the active sites ( free
active sites more likely to be saturated )
Make it easier for the substrate to enter the active site .

By making the shape of active site more

complemnatry
 1.V max………all active sites are occupied / enzyme
5. Enzyme inhibitors : concentration
2. Km …measure the affinity of the enzyme to substrate …the
A) competitive inhibitor higher the affinity ( lower Km ) , less substrate needed to reach
V max / saturate the active sites
1. Inhibitor has similar structure to the substrate , which is
complementary to the active site . V max -> Enzym

. The inhibitor compete with substrate on the active site Km

3. So the inhibitor bind to the active site , substrate can’t bind to
active site .
4. So the frequency of successful collision decrease, so fewer
ESCs
5. So at lower substrate concentration , so slower rate of
reaction .
6. So higher substrate concentration is needed to reach the V
max .
Effect on Vmax and Km
V max ..remain the unchanged ….where the inhibitor compete in
the active site , so reducing frequency of successful collisions , so
fewer ESCc .. thus upon increasing substrate concentration , the
inhibitor will have least effect , so V max remains the same . ..

Km ….value of km will, increase …..as there is a decrease in the affinity of the enzyme to the substrate , as
the competitive inhibitor compete on the active site with substrate , so higher substrate concentration is
needed to reach V max .

B) non competitive inhibitor p1

1. Inhibitor binds to a site other than the active p2

site /allosteric site .

2. This will disrupt the arrangement of
hydrogen bonds and hydrophobic interactions
holding the enzyme in a specific 3D shape
…….causing a change in the tertiary structure
…change in shape if active site
3. Substrate is unable to bind to the active site
to form ECS…so fewer ECSc
4. V max …is not reached even by increasing
substrate concentration.
But km remains the same . .

V max …..all active sites are occupied / enzyme concentration

Km ….measures affinity of enzyme to substrate
 V max …..all active sites are occupied / enzyme concentration
V max decrease ; Km ….measures affinity of enzyme to substrate

V max depends on enzyme concentration

Where the non competitive inhibitor binds to allosteric site causing a change in shape of active site
Preventing substrate from binding to the active site , thus decreasing enzyme activity
And upon increasing substrate concentration will still show no effect

Km
Remains the same
As the inhibitor doesn’t comepete on the active site
So no change in the affinity of enzyme to the substrate

Only one female

Competitive Non Competitive

Compete on active site Bind to allosteric site…change shape

of active site

V max Remain the same Decreased

Km Change ( increase ) affinity decrease Remain the same

Example of a non competitive inhibitor

In many situations inhibition is essentials …in metabolic reactions …where we need end product inhibition
To maintain balance and allowe effecient metabolism
 Advantages of using immobilised enzymes

1. Stability of enzyme to any change in temperature ' ( thermostability ) and any

change in pH …allowing enzymes to have longer shelf life
2. Reducing end product inhibition
&
3. Recycled ( used many times )
4. Product not contaminated with enzymes
&
5. Allow large scale production exposing substrate to high enzyme concentration
&
so more product per unit time

Advantages of using smaller beads

1. Large surface area to volume ration

2. Pack more beads into column
3. Slow down passage of substrate through the column
Allowing more time for the enzyme to be exposed to substrate
So faster rate of reaction