Transcript Part 2

Enzymes: Catalytic strategies &
regulatory mechanisms
Binding of a substrate to an enzyme catalytic site brings
about conformational changes in the enzyme with
multiple weak interactions being formed with the
substrate molecule. The amino acid side chains of the
enzyme aid in cleavage and formation of bonds thereby
providing a variety of mechanisms for catalysis to occur.
Harini Chandra
Master Layout (Part 1)
1
This animation consists of 4 parts:
Part 1 – Covalent catalysis
Part 2 – Acid-base catalysis
Part 3 – Metal ion catalysis
Part 4 – Regulatory strategies
Nucleophilic group
2
3
X
A
B
A
B
X
Substrate
Covalent
bond
H2O
4
A
5
Product
Source: Modified from Biochemistry by A.L.Lehninger et al., 4 th edition (ebook)
X
B
Product
1
2
Definitions of the components:
Part 1 – Covalent catalysis
1. Covalent catalysis: A catalytic strategy wherein the active site of an enzyme is lined
with a reactive group, usually a powerful nucleophile, that undergoes temporary
covalent modification during the course of the reaction. A transient covalent bond
between the enzyme and substrate facilitates catalysis by providing a pathway with
lower activation energy.
2. Substrate: The molecule(s) present at the beginning of a reaction that are modified
by enzyme are known as substrate(s). An enzymatic reaction may have one or more
substrates depending upon the reaction.
3
3. Covalent bond: A chemical bond formed between atoms of two reacting species
that involves the sharing of outer orbital electrons for bond formation.
4. Nucleophilic group: An electron rich group that readily attacks a positively charged
centre to form a chemical bond by donating its electrons. The term nucleophile
translates to “nucleus lover”.
4
5
5. Product: The molecules produced as a result of an enzymatic reaction are known as
the products. A reaction may yield one or more products with the enzyme being
regenerated at the end of the reaction.
Part
1,
Step
1:
1 Covalent catalysis
Nucleophilic group
H2O
X
Regenerated
catalyst
2
A
3
4
Products
Substrate
Covalent
bond
Action Description of the action
As
shown in
animatio
n.
5
B
First show ‘A’ and ‘B’ joined by thick line as shown along
with ‘X’. ‘X’ must be shown to attack ‘A’ and when this
happens, the line between A&B must become a dotted line
and a new dotted line must appear between A&X. Next, B
must move away and the dotted line must disappear while
the dotted line between A&X must become a thick line. Next
the blue ‘H2O’ must appear and must pass through the line
between A&X. When this happens, the line between A&X
must break and they must move apart from each other.
Audio Narration
Covalent catalysis involves the formation of a transient
covalent bond between the nucleophile present in the
enzyme and the substrate molecule. Formation of this
bond provides an alternative reaction pathway that has
lower activation energy than the uncatalyzed reaction.
Several amino acid side chains act as effective
nucleophiles that facilitate the reaction. The enzyme is
regenerated in its unaltered form at the end of the
reaction.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
1,
Step
2:
1
Example for covalent catalysis - Chymotrypsin
O
OH
2
X
O
C
R
Ser-195
C
O
Substrate
XH
Enzyme
3
H2O
O
OH
HO
Ser-195
Regenerated
enzyme
4
Action Description of the action
As
shown in
animatio
n.
5
R
Ser-195
First show the ‘enzyme’ and ‘substrate’. Then show
the black arrow appearing after which the grey arrow
must appear followed by removal of the group ‘XH’
as shown and appearance of the figure after the
arrow. The red line shown must then appear. The
blue oval marked ‘H2O’ must then appear followed
by the black arrow as shown. This must be followed
by appearance of the downward grey arrow and the
figures shown below it.
C
R
Product
Audio Narration
Chymotrypsin is one such enzyme that carries out catalysis by
covalent modification. It possess a catalytic triad of histidine, aspartic
acid and serine at its active site with the serine at position 195 serving
as a highly powerful nucleophile. The reaction between the serine
hydroxyl group and the unreactive carbonyl group of the substrate
helps in bringing about product formation with regeneration of the
enzyme after the reaction. Covalent modification of this serine residue
led to irreversible inactivation of the enzyme, which clearly suggested
that it performs a vital role in catalysis.
Source: Modified from Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Master Layout (Part 2)
1
This animation consists of 4 parts:
Part 1 – Covalent catalysis
Part 2 – Acid-base catalysis
Part 3 – Metal ion catalysis
Part 4 – Regulatory strategies
R1
R
OH
R1
2
C
O
N
R2
Substrates
R
O
C
H
O
Products
R1
H
N
3
R2
R
C
O
H
H
O
N
H
Base
4
5
O
H
C
N
R1
O
R
O
C
N
R2
BH
H
Intermediate
B
R1
R
R2
H
O
R2
1
2
Definitions of the components:
Part 2 – Acid-base catalysis
1. Acid-base catalysis: Biochemical reactions involving the formation of unstable
charged intermediates are often stabilized by transfer of protons to or from the substrate
or intermediate. In case of enzyme catalyzed reactions, weak proton donors or acceptors
are often present as amino acid side chains at the active site of the enzyme itself. These
groups mediate proton transfer reactions which provide rate enhancement of several
orders of magnitude.
2. Substrate(s): The molecules present at the beginning of a reaction that are modified
by means of the enzyme are known as substrates. An enzymatic reaction may have one
or more substrates depending upon the reaction.
3
4
5
3. Enzyme: The biocatalyst responsible for bringing about an increase in the rate of
reaction for conversion of substrate to product.
4. Intermediate: Enzymatic reactions proceed through formation of a transition state i.e.
an intermediate state in between substrate and product having higher free energy than
that of either the substrate or product. The transition state is the least stable species of
the reaction pathway due to its high free energy and is therefore the most seldom
occupied.
5. Base: An aqueous solution or substance that can accept hydronium ions by donating
its free electron pair is known as a base.
6. Product(s): The molecules produced as a result of an enzymatic reaction are known
as the products. A reaction may yield one or more products with the enzyme being
regenerated at the end of the reaction.
Part
2,
Step
1:
1
Acid-base catalysis
R
R1
OH
C
O
N
R2
2
H
Substrates
3
R1
R
O
H
C
N
H
4
O
R2
Intermediate
Action Description of the action
As
shown in
animatio
n.
5
First show the two ‘substrates’ on top.
The green circle containing ‘OH’ group
must move towards the yellow circle
containing ‘C’ as shown. The down arrow
must then appear to give the figure
below.
Audio Narration
Biochemical reactions involving the formation of
unstable charged intermediates are often
stabilized by transfer of protons to or from the
substrate or intermediate. For non-enzymatic
reactions, acid-base catalysis may involve only
the hydronium or hydroxyl ions of water, referred
to as specific acid-base catalysis.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
2,
Step
2:
1
Acid-base catalysis
R1
R
2
Base
C
O
H
O
N
B
R2
H
3
R1
R
O
BH
C
N
R2
H
4
Action Description of the action
As
shown in
animatio
n.
5
O
Next, show the blue circle ‘base’
appearing which must move towards the
green circle containing ’OH’ as depicted. It
must then remove the red ‘H’ from it and
the figure below must appear. The blue
circle must then move away.
Audio Narration
In many cases, however, water alone does not
suffice to catalyze the reaction. In such cases,
proton transfer is facilitated by weak organic
acids or bases. Organic acids act as proton
donors while organic bases can serve as proton
acceptors.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
2,
Step
3:
1
Acid-base catalysis
R1
R
2
C
O
O
N
BH
R2
H
3
R1
R
O
H
B
4
C
N
5
Next, the blue circle must move towards the
pink circle containing ‘N’. The red ‘H’ must
then be transferred to this group as shown in
the figure below. The blue circle must then
move away.
R2
H
Action Description of the action
As
shown in
animatio
n.
O
Audio Narration
In case of enzyme catalyzed reactions, weak
proton donors or acceptors are often present as
amino acid side chains at the active site of the
enzyme itself. The precise positioning of these
groups within the active site mediate proton
transfer reactions which can provide rate
enhancements of several orders of magnitude.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
2,
Step
4:
1
Acid-base catalysis
R1
R
O
H
2
C
O
N
R2
H
3
R1
H
R
O
C
O
R2
H
Products
4
Action Description of the action
As
Show the arrows appearing in the
shown in figure above at the positions
animatio indicated followed by the downward
n.
5
N
arrow and the ‘products’ below.
Audio Narration
Acid-base catalysis is a common mechanism of
action employed by many enzymes. It is often used
in combination with another mechanism such as
covalent catalysis. The ease of stabilization of
charged intermediates by the amino acid side chains
helps in lowering the activation energy for product
formation.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
2,
Step
5:
1
Example for acid-base catalysis - Chymotrypsin
Active site – catalytic triad
His
Ser
Asp
57
195
102
2
Substrate
Chymotrypsin enzyme
Oxyanion
hole
3
4
Tetrahedral intermediate
Action Description of the action
As
shown in
animatio
n.
5
(PLEASE RE-DRAW ALL FIGURES.)
First show the top left figure appearing with
its labels followed by appearance of the
arrow and the compound above it. Next the
second figure on the right must appear
followed by the downward arrow and the
figure below it.
Source: Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Audio Narration
Chymotrypsin is one such enzyme that employs both
covalent catalysis as well as acid-base mechanism. The
arrangement of the catalytic triad, consisting of Aspartic
acid, Histidine and Serine, at the enzyme’s active site is
such that the histidine residue serves as a general base
catalyst by polarizing the hydroxyl group of serine. The
alkoxide ion thus generated in the serine residue makes it
a more powerful nucleophile.
Part
2,
Step
6:
1
Example for acid-base catalysis - Chymotrypsin
Acyl-enzyme intermediate
2
Tetrahedral intermediate
Product 1
3
4
Acyl-enzyme intermediate
Action Description of the action
As
shown in
animatio
n.
5
(PLEASE RE-DRAW ALL FIGURES.)
In continuation with the previous slide. The
grey arrow must then appear after
‘tetrahedral intermediate’ followed by the
figure on the right. Next the arrow must
appear along with the curved arrow showing
removal of ‘product 1’ followed by the figure
below.
Audio Narration
Following substrate binding and nucleophilic attack of the serine on
the carbonyl group, the geometry of the intermediate becomes
tetrahedral and the negative charge developed on the carbonyl
oxygen gets stabilized through interactions with other side chains
of the proteins, in a site known as the oxyanion hole. An internal
proton transfer then causes the tetrahedral intermediate to collapse
and generate the acyl-enzyme intermediate after which the amine
group is released from the active site.
Source: Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Part
2,
Step
7:
1
Example for acid-base catalysis - Chymotrypsin
Acyl-enzyme intermediate
2
Acyl-enzyme intermediate
Oxyanion
hole
3
4
Tetrahedral intermediate
Action Description of the action
As
shown in
animatio
n.
5
(PLEASE RE-DRAW ALL FIGURES.)
In continuation with the previous slide.
The reaction arrow must appear along
with entrance of a molecule of ‘H2O’ as
shown. This is followed by appearance of
the figure on the right after which the
downward arrow must appear along with
the figure below.
Audio Narration
Once the amine group leaves the enzyme’s active site, it is
replaced by a molecule of water which carries out
hydrolysis of the ester group of the acyl-enzyme
intermediate. Mechanism for hydrolysis again proceeds via
formation of a tetrahedral intermediate with histidine acting
as a general acid catalyst and the negative charge on
oxygen being stabilized by residues in the oxyanion hole.
Source: Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Part
2,
Step
8:
1
Example for acid-base catalysis - Chymotrypsin
2
Tetrahedral intermediate
Product 2
3
4
Regenerated enzyme
Action Description of the action
As
shown in
animatio
n.
5
(PLEASE RE-DRAW ALL FIGURES.)
In continuation with the previous slide. The
arrow must then appear to show the figure
on the right top followed by the downward
arrow and removal of the compound shown
as ‘product 2’ followed by the appearance
of the figure below.
Audio Narration
The tetrahedral intermediate then breaks down to
liberate the second product in the form of a
carboxylic acid along with regeneration of the
enzyme which is then ready for another round of
catalysis. Internal proton transfers between
amino acid side chains therefore play a vital role
in acid-base catalysis by enzymes.
Source: Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Master Layout (Part 3)
1
This animation consists of 4 parts:
Part 1 – Covalent catalysis
Part 2 – Acid-base catalysis
Part 3 – Metal ion catalysis
Part 4 – Regulatory strategies
R1
H
2
H
M2+
O
M2+
H
Metal ion
C
O
H
O
Substrate
R2
3
R1
4
M2+
O
5
Regenerated
catalyst
C
R1
H2O
O
M2+
H
R2
Product
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
C
O
H
R2
Intermediate
O
1
2
Definitions of the components:
Part 3 – Metal ion catalysis
1. Metal-ion catalysis: Metal ions, either present in solution or bound to the enzyme
itself, facilitate catalysis by forming favorable interactions that orient the substrate and
enzyme in suitable positions for transition state, and subsequently, product formation.
These interactions help in stabilizing the intermediates formed, thereby lowering the
activation energy required for reaction to occur.
2. Substrate(s): The molecules present at the beginning of a reaction that are
modified by means of the enzyme are known as substrates. An enzymatic reaction
may have one or more substrates depending upon the reaction.
3
3. Enzyme: The biocatalyst responsible for bringing about an increase in the rate of
reaction for conversion of substrate to product.
4. Intermediate: Enzymatic reactions proceed through formation of a transition state
i.e. an intermediate state in between substrate and product having higher free energy
than that of either the substrate or product. The transition state is the least stable
species of the reaction pathway due to its high free energy and is therefore the most
seldom occupied.
4
5
5. Metal ion: Metals usually present in their divalent state (i.e. +2 charge) facilitate
catalysis. One or more metal ion has been found to be required for catalysis of nearly
one third of all enzymatic reactions. One of the most commonly found metal ions in
enzymatic mechanisms is Zn2+.
6. Product(s): The molecules produced as a result of an enzymatic reaction are
known as the products. A reaction may yield one or more products with the enzyme
being regenerated at the end of the reaction.
Part
3,
Step
1:
1
Metal ion catalysis
2
H
M2+
4
M2+
O
Metal ion
3
O
H
H
Water
Strong nucleophile
generated
Action Description of the action
First show the purple oval along with the
As
shown in ‘water’ figure. The purple circle must then
animatio approach the blue circle of water. When
n.
5
H
this happens, the red line must appear
between the two and one green ‘H’ must
leave in the form of ‘H+’ as depicted to
give the figure on the right.
Audio Narration
Metal ions, either present in solution or bound to the
enzyme itself, facilitate catalysis by forming favorable
interactions between enzyme and substrate or in the
transition state. Metal ions in the active site of the
enzyme typically react with a water molecule and
activate it by facilitating generation of a strong
nucleophile in the form of a hydroxide ion.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
3,
Step
2:
1
Metal ion catalysis
R1
R1
2
M2+
O
C
M2+
O
C
O
O
H
H
3
4
Strong nucleophile
generated
Intermediate
Substrate
Action
As
shown in
animatio
n.
5
R2
R2
Description of the action
In continuation with previous slide.
Show the ‘substrate’ molecule
appearing after which the black arrows
must appear as shown. The grey arrow
must then appear along with the figure
on the right.
Audio Narration
The nucleophilic alkoxide ion attacks the
unreactive carbonyl group to form a tetrahedral
intermediate in which the charges are stabilized by
the metal ion. These favorable interactions help in
orienting the substrate and enzyme in suitable
positions for transition state, and subsequently,
product formation.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
3,
Step
3:
1
Metal ion catalysis
H2O
R1
H2O
2
R1
O
C
O
O
M2+
3
M2+
Regenerated
catalyst
Intermediate
4
Action Description of the action
In continuation with previous slide.
As
shown in Show the blue ‘H2O’ appearing which
animatio must move towards the violet oval as
n.
5
shown. This is followed by appearance
of the small black arrow after which the
grey arrow and the figures on the right
must appear as depicted.
O
H
H
R2
C
R2
Product
Audio Narration
Hydrolysis of the stabilized intermediate leads to
product formation along with liberation of the
regenerated metal catalyst. Thus by lowering the
activation energy of the transition state, metal ions
facilitate enzyme catalyzed reactions. Almost one
third of known enzymes have been found to
require one or more metal ions for their activity.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
3,
Step
4:
1
Example for metal ion catalysis – Carbonic anhydrase
2
His
119
His
96
3
4
H
Zn2+
His
94
H
Active site
His
119
His
96
O
O
H
Strong nucleophile
generated
Carbonic
anhydrase
Action Description of the action
black arrow mark as shown after which
the grey arrows, the figure on the right
and the green ‘H+’ must appear.
Zn2+
His
94
Water
First show appearance of ‘carbonic
As
shown in anhydrase’, ‘active site’ and ‘water’.
animatio This is followed by appearance of the
n.
5
H
Audio Narration
Carbonic anhydrase is an enzyme responsible for
hydration and dehydration reactions of carbon dioxide and
bicarbonate respectively and has been found to have a
divalent zinc ion associated with its activity. The zinc ion in
its active site is bound to the imidazole rings of three
histidine residues as well as to a molecule of water. This
binding to water facilitates formation of the hydroxide
nucelophile with concomitant release of a proton.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
3,
Step
5:
1
Example for metal ion catalysis – Carbonic anhydrase
O
His
119
2
3
His
96
Zn2+
H
His
94
O
Substrate
His
119
His
96
4
C
O
His
94
O
Zn2+
O
C
H
Intermediate
O
Action Description of the action
5
Electrostatic
stabilization forces
Audio Narration
The generated hydroxide ion at the active site
then attacks the carbon dioxide substrate,
converting it into a bicarbonate ion. The negative
charge generated on the oxygen atom is
This is followed by the grey downward arrow
and the figure show below. The dotted red line stabilized by interactions with the zinc ion.
must appear in the end after the figure is
shown as depicted in animation.
In continuation with previous slide.
As
shown in The ‘substrate’ should then appear after which
animatio the black arrows must be shown as depicted.
n.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
3,
Step
6:
1
Example for metal ion catalysis – Carbonic anhydrase
H
His
119
O
2
H
His
119
His
96
3
His
96
His
94
O
Zn2+
O
C
H
O
His
94
O
Zn2+
Regenerated
enzyme
O
O
C
O
H
Product
(bicarbonate)
4
Action Description of the action
5
H
In continuation with previous slide.
As
shown in Another ‘water’ molecule shown above
animatio must then appear and approach the
n.
brown oval. The grey arrow must then
appear followed by the figures shown on
the right.
Audio Narration
Binding of another molecule of water to the zinc
ion at the active site of the enzyme leads to
release of the bicarbonate ion and regeneration
of the enzyme molecule ready for another round
of catalysis. Several pH related studies have
provided substantial proof for this mechanism.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
H
Master Layout (Part 4)
1
This animation consists of 4 parts:
Part 1 – Covalent catalysis
Part 2 – Acid-base catalysis
Part 3 – Metal ion catalysis
Part 4 – Regulatory strategies
Enzyme regulatory
strategies
2
3
4
5
Allosteric/
feedback
inhibition
Isozyme
forms
Reversible
covalent
modification
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Proteolytic
activation
1
2
3
4
5
Definitions of the components:
Part 4 – Regulatory strategies
1. Allosteric/feedback inhibition: Enzymes that contain distinct catalytic and
regulatory sites are said to be allosteric in nature. Binding of regulatory molecules at
the regulatory sites triggers off a series of conformational changes that ultimately reach
the active site and prevents binding of substrate molecule thereby regulating catalysis.
In many pathways, the reaction that is regulated involves the enzyme catalyzing the
first step of the pathway so that all further reactions taking place after the first also
automatically get regulated due to lack of substrate availability. When this enzyme is
regulated by the final product of the reaction pathway, it is known as feedback
inhibition. Most allosteric enzymes are subject to feedback control, a popular example
being aspartate transcarbamoylase.
2. Isozyme forms: Isoenzymes are homologous enzymes within a single organism
that differ slightly in their structure but catalyze the same reaction. They are mostly
expressed in different tissues and have differing kinetic parameters such as substrate
affinity (Km) and maximum velocity (Vmax). These differing properties allow the
isozyme forms to be differentially regulated at varying points of time. Example: Lactate
dehydrogenase exists in different forms in heart and muscle tissue. The heart isozyme
has a higher affinity for substrates and is allosterically inhibited by high levels of
pyruvate while the muscle enzyme is not.
3. Reversible covalent modification: Several covalent modifications such as
phosphorylation, uridylylation, methylation etc. significantly alter the catalytic activity of
enzymes. Covalently modified enzymes may either have increased or decreased
activity for the reaction it catalyzes. The enzymes responsible for covalent modification
of other enzymes are usually regulated by further control mechanisms. Example:
Enzymes of glycogen metabolism such as glycogen phosphorylase are regulated by
covalent modification.
1
2
3
4
5
Definitions of the components:
Part 4 – Regulatory strategies
4. Proteolytic activation: Many enzymes exist in their inactive forms, known as
zymogens, and need to be activated by hydrolysis of one of more peptide bonds. This
removal of certain regulatory residues irreversibly converts the enzyme into its active
form. The enzyme is then degraded after completion of catalysis. Example: Digestive
enzymes such as trypsin, chymotrypsin as well as blood clotting factors are regulated
by mechanism of proteolytic activation.
Part
4,
Step
1:
1
Enzyme regulatory
strategies
2
3
Allosteric/
feedback
inhibition
Isozyme
forms
Reversible
covalent
modification
Proteolytic
activation
4
Action Description of the action
As
shown in
animatio
n.
5
(Please redraw figures.)
The top heading must be shown followed by the four
arrows and the headings in the circles below. The user
should be allowed to click on any of the headings in the
circles to view the description provided in the previous two
slides. Another ‘proceed’ tab must be present on the right
bottom and when user clicks on that, the text in the blue
circle and yellow circle must be highlighted after which it
must move to the next slide.
Audio Narration
Activity of all enzymes must be regulated to ensure that they
function only to the desired extent at the appropriate locations
within an organism. Common mechanisms of regulation include
allosteric or feedback inhibition, control of isozyme forms,
reversible covalent modification and proteolytic activation. <If user
clicks on any of these four tabs, the description as given in
previous two slides must be narrated.>
Source: Modified from Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Part
4,
Step
2:
1
Allosteric/feedback inhibition
Substrate B
2
3
Enzyme C
Enzyme D
Substrate E
Enzyme A
Allosteric enzymes DO NOT obey
Michaelis-Menten kinetics.
Action Description of the action
As
shown in
animatio
n.
5
Substrate D
Substrate A
Enzyme B
4
Substrate C
First show blue ‘enzyme A’ along with green ‘substrate A’. Green
substrate must bind in the pocket as shown after which its shape
must change to a brown oval. This oval must leave the pocket
after which the sequence of arrows and the other shapes must
appear as depicted in animation. When ‘Substrate E’ appears, it
must move towards ‘enzyme A’ and bind in the other pocket as
depicted. When this happens, the pocket on top must change
shape as shown and the green substrate should not be able to
bind to it. Then red crosses must appear across all the other
reaction arrows as well.
Audio Narration
Allosteric enzymes, which possess distinct regulatory and
catalytic sites, are often found as the first enzyme of a reaction
pathway. Regulation of the first enzyme of a pathway by the final
product of the pathway is known as feedback inhibition. Binding
of the regulator molecule to the regulatory site of the enzyme
triggers a series of conformational changes that are ultimately
transmitted to the active site where substrate binding is then
inhibited. It has been observed that allosteric enzymes do not
obey regular Michaelis-Menten kinetics.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
4,
Step
3:
1
Example for allosteric/feedback inhibition – Aspartate Transcarbamoylase
Catalytic subunits
Regulatory subunits
2
Inhibitor
CTP
Substrate
PALA
Enzyme
3
PALA
CTP
CTP
CTP
CTP
PALA
4
Stabilized R state
Stabilized T state
Action Description of the action
As
shown in
animatio
n.
5
PLEASE REDRAW ALL FIGURES.
First show the figure on top with its labels.
Next show the left arrow appearing with
the violet circles binding to the red circles
as shown. Next show the right arrow
appearing with blue circles binding to the
yellow regions as shown.
Audio Narration
Aspartate transcarbamoylase, which catalyzes the first step of pyrimidine
biosynthesis, is an allosteric enzyme having distinct regulatory and
catalytic subunits. Binding of substrate to the catalytic subunits induces
conformational changes that stabilize the relaxed state or R state of the
enzyme, thereby facilitating the enzymatic reaction. The inhibitor for this
enzyme is CTP which is the final product of the pathway. Binding of
inhibitor to the regulatory subunits stabilizes the tense state or T state of
the enzyme thereby preventing the reaction from taking place.
Source: Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Part
4,
Step
4:
1
Isozyme forms
Different regulatory molecules
Substrate
Substrate
Different
regulatory site
2
3
Tissue A –
isozyme A
Similar catalytic
site
Tissue B –
isozyme B
Same reaction catalyzed –
different
Product kinetic parameters
such as Vmax, Km.
Product
4
Action Description of the action
As
shown in
animatio
n.
5
First show the green & brown ‘isozymes A&B’ appearing. The
arrows with labels must appear, flash a couple of times and then
disappear. Next, the ‘substrate’ must appear, enter the ‘catalytic
site’ as shown where it must change shape and then leave as the
‘product’. This must take place simultaneously for both A&B. The
reaction taking place in B must be faster than A as depicted in
animation. Once this happens, the text message shown below in
red must appear. Next the violet pentagon must bind to the
‘regulatory site’ on top in B but not in A as depicted. This is
followed by text message on top.
Audio Narration
Isoenzymes are homologous enzymes within a single organism
that differ slightly in their amino acid sequence but catalyze the
same reaction. These enzymes are mostly expressed in different
tissues and have differing kinetic parameters such as substrate
affinity (Km) and maximum velocity (Vmax). They may also
have different regulator molecules that allow them to be
differentially expressed and regulated depending on the
requirement for that particular tissue.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
4,
Step
5:
1
Example for isozyme regulation – Lactate dehydrogenase
MUSCLE ISOZYME – M4
HEART ISOZYME – H4
Lactate
2
3
High affinity for
substrate
LDH
LDH
Low affinity for
substrate
Not inhibited by
pyruvate
Inhibited by pyruvate
Pyruvate
4
Action Description of the action
As
shown in
animatio
n.
5
PLEASE REDRAW ALL FIGURES.
First show the figure of heart and muscle
along with the ‘LDH’ enzyme. Next show
the reaction in the centre followed by the
text ‘high affinity…’. Next show the blue
pentagon binding to the pocket on the left
but being unable to bind on the right
followed by the text below as depicted.
Audio Narration
Lactate dehydrogenase is an enzyme involved in anaerobic glucose
metabolism that is present as two isozyme forms in human beings.
The tetrameric heart enzyme , which requires an aerobic environment
to function, has higher affinity for its substrate than the muscle
enzyme. Despite having 75% sequence homology, they also differ in
that high levels of pyruvate allosterically inhibit the heart enzyme but
not the muscle form.
Source: Modified from Biochemistry by Lubert Stryer et al., 6th edition (ebook)
Part
4,
Step
6:
1
Reversible covalent modification
ATP
ADP
Substrate
P
2
Substrate
Kinase
Enzyme
Enzyme
Less Active
3
Phosphatase
Pi
More Active
H2O
Product
Product
4
Action Description of the action
As
shown in
animatio
n.
5
First show reaction on the left appearing along
with the ‘less active enzyme’. A red cross must
appear over the reaction arrow. The curved
arrows towards the right must then appear
followed by the ‘more active enzyme’ on the right.
When this appears, the reaction must take place
as shown on right. Finally the curved arrows
towards the left must appear as depicted.
Audio Narration
Reversible covalent modification is another commonly employed enzyme
regulatory strategy. The most widely observed modification is
phosphorylation, which is carried out by various enzyme kinases with the
help of ATP as a phosphoryl donor. Some enzymes are more active in
their phosphorylated forms while others are less active in this form.
Dephosphorylation is carried out by the phosphatase enzyme. Enzymes
involved in glycogen metabolism are regulated by reversible
phosphorylation.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
4,
Step
7:
1
Reversible covalent modification
2
3
Type of covalent
modification
Amino acid
modified
Donating group
Phosphorylation
Ser, Thr, Tyr His
ATP, GTP
Adenylylation
Tyr
ATP
Uridylylation
Tyr
UTP
ADP-ribosylation
Arg, Gln, Cys
NAD
Methylation
Glu
S-adenosyl
methionine
4
Action Description of the action
As
shown in
animatio
n.
5
The rows of the table must appear one at a time.
Audio Narration
Apart from phosphorylation, which most commonly takes place at
serine, threonine and tyrosine residues, other reversible covalent
modifications include adenylylation, uridylylation, methylation and
ADP-ribosylation which modify different amino acid residues of the
proteins.
Source: Modified from Biochemistry by A.L.Lehninger et al., 4th edition (ebook)
Part
4,
Step
8:
1
Proteolytic activation
Enzymeactive
inactive
Enzyme
form
form (zymogen)
2
Protease
3
Substrate
Product
4
Action Description of the action
As
shown in
animatio
n.
5
First show the reaction coming in as depicted
followed by the ‘inactive’ enzyme’ appearing
on the arrow followed by the red cross. Next
show the pie shaped object ‘protease’
entering which must cleave the red portion
and remove it. The red cross must then
disappear and the label for enzyme must
change as shown.
Audio Narration
Several enzymes exist in their inactive forms, known as zymogens,
where they do not possess any catalytic activity. In order to become
active, they need to be activated by hydrolysis of one of more
peptide bonds by various protases. This removal of certain
regulatory residues irreversibly converts the enzyme into its active
form. Unlike reversible modification, the enzyme is degraded after
completion of catalysis.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Part
4,
Step
9:
1
Example for proteolytic activation – Chymotrypsin
Chymotrypsinogen
(inactive)
2
1
1
15 16
3
Pi-chymotrypsin
Substrate
Product
Substrate
Product
245
Pi-chymotrypsin
1
4
245
Trypsin
13 16
A-chain
245
146 149
B-chain
C-chain
a-chymotrypsin (active)
Action Description of the action
As
shown in
animatio
n.
5
First show the ‘inactive chymotrypsinogen’ above
followed by appearance of the reaction on the left
with the red cross on it. Next show the ‘trypsin’
which must cleave the blue rod as shown to give
‘pi-chymotrypsin’. The green ‘trypsin’ must again
cleave the blue chain to give the figure below of
alpha-chymotrypsin followed by the reaction
sequence on the right as depicted.
Audio Narration
Several digestive enzymes as well as clotting factors are regulated by
proteolytic activation. Chymotrypsin, a digestive enzyme that hydrolyzes
proteins in the small intestine, exists in its zymogen form within membrane
bound granules after synthesis in the acinar cells of the pancreas. The
proteolytic enzyme trypsin converts it into its fully active form by cleavage of a
peptide bond between arginine at position 15 and isoleucine at position 16.
The resulting enzyme known as pi-chymotrypsin is acted upon by other such
molecules to yield the completely active and stable alpha-chymotrypsin which
consists of three chains linked by inter-chain disuphide bonds.
Source: Modified from Biochemistry by Lubert Stryer et al., 6 th edition (ebook)
Interactivity
option
1:Step
No:1
1
Match the following enzymes with their appropriate regulatory strategies.
Enzyme name
2
3
Regulatory mechanism
A.
Glycogen phosphorylase
1. Isozyme forms
B.
Aspartate transcarbamoylase
2. Covalent modification
C.
Chymotrypsin
3. Allosteric inhibition
D.
Lactate dehydrogenase
4. Proteolytic activation
4
Interacativity Type
Match the following.
5
Options
User must be
allowed to drag the
A, B, C, D into the
dotted boxes shown
on the right.
Results
User must drag & drop the A, B, C, D provided in the
left column into the dotted boxes present in the right
column. Every time the user drags and matches it
correctly, that box must then turn green. If user gets it
wrong, then it must move back automatically into the
left column. Correct answers are 1-D, 2-A, 3-B and 4C.
1
2
Questionnaire
1. Which of the following amino acid residues is not part of the catalytic triad of the active site
of chymotrypsin?
Answers: a) His b) Asp c) Ser d) Pro
2. Which metal ion bound to the enzyme carbonic anhydrase is responsible for its activity?
3
Answers: a) Zn2+ b) Fe2+ c) Mg2+ d) Mn2+
3. The attachment of a metal ion to water generates which of the following molecules?
Answers: a) Strong electrophile b) Strong nucleophile c) Weak electrophile d) Weak
nucleophile
4
4. S-adenosyl methionine is the donor group for which type of reversible covalent
modification?
Answers: a) Phosphorylation b) Adenylylation c) Methylation d) UDP-ribosylation
5
Links for further reading
Books:
Biochemistry by Stryer et al., 5th & 6th edition
Biochemistry by A.L.Lehninger et al., 4th edition
Biochemistry by Voet & Voet, 3rd edition
Fundamentals of Enzymology by Price & Stevens, 3rd
edition