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CHMI 2227E
Biochemistry I
Enzymes:
-
Regulation
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity


In any organism, enzymes are always kept in check so that their activity can
match the needs of the cell:

Enzymes can be activated: the reaction is stimulated to generate more product;

Enzymes can be inactivated (or inhibited): the reaction is slowed down to
decrease the amount of product;
Several strategies are used to modulate (i.e. activate or inhibit) enzymes:





1-Allostery
Inhibition by product
Activation by
substrate/cofactor
2- Binding of regulatory
subunits
3- Covalent modification:

Phosphorylation on Ser/Thr/Tyr

4- Degradation of the enzyme

5- Limited proteolysis
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
1. Allostery

Widely used in metabolic enzymes:
 Inhibition
by the end product of a pathway;
 Activation
by a product generated early on in the
pathway;

Based on the principle of cooperativity:
 The
binding of a small molecule to the enzyme
modifies the 3-D structure of the protein and alters its
ability to catalyse the reaction;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
1. Allostery

Example: Aspartate transcarbomoylase (ATCase):

Involved in the first of a series of reactions leading to the production of CTP;

CTP (the end product) inhibits ATCase by allostery;

ATP activates ATCase, also by allostery (competes with CTP for binding
regulatory sites on ATCase);
ATCase
Carbomoyl
phosphate
Asp
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
1. Allostery - ATCase
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
1. Allostery - ATCase
CTP
ATCase
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
2. Regulatory subunits
cAMP is produced from ATP by the action of
adenylate cyclase;

The binding of cAMP to the regulatory
subunit of PKA frees the catalytic subunits,
which are now fully active;
ATP
cAMP
Caffeine
AMP
CHMI 2227 - E.R. Gauthier, Ph.D.
cAMP
Phosphodiesterase
Adenylate
Cyclase

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Regulation of enzyme activity
3. Regulation by covalent modification

Specific amino acid side chains of several
enzymes are the target of covalent modifications
(catalysed by yet other enzymes…);
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
3. Regulation by phosphorylation

The addition of a
phosphate group
(phosphorylation) by
protein kinases and their
removal (by protein
phosphatases) is
frequently used to
modulate the activity of
enzymes;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
3. Phosphorylation  Adrenaline
Adrenaline receptor
Adenylate cyclase
Adrenaline
Outside the cell
G Protein
g
b
a
g
↑Energy
Run Like Hell!
a
Inside the cell
a
cAMP
Glycogen
(glucose stores)
↑Intracellular glucose
b
Glycogen
Phosphorylase-PO4
Phosphorylase
kinase
ATP
Protein Kinase A
(inactive)
PKA-cAMP
(active)
Phosphorylase
Kinase-PO4
Glycogen
CHMI 2227 - E.R. Gauthier, Ph.D.
Phosphorylase
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Regulation of enzyme activity
4. Regulation of enzyme stability

Proteins are constantly being made (i.e. synthesized)
and destroyed (i.e. degraded);

The tight regulation of protein synthesis and degradation
participates to the regulation of enzyme activity;
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
4. Regulation of enzyme stability
Ub

E1
 76
amino acid protein
 Tags other proteins for
degradation;
Ub
Enzyme
E1
E2
Ub
E2
E3
Ub
E3
Ubiquitin:

Ubiquitin is attached
to other proteins by a
series of 3 enzymes
(E1, E2 and E3);
Enzyme
Ub
Ub
Ub
Ub
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
4. Regulation of enzyme stability

Polyubiquitylated proteins are
targeted to a HUGE protein complex
called the proteasome:



Ub
Ub
Ub
Ub
Ub
Ub
The degradation of the enzyme will
result in a decrease in the amount of
the product of the reaction catalysed
by the enzyme (well, duh!);
CHMI 2227 - E.R. Gauthier, Ph.D.
Ub
Ub
Contains several subunits with
protease (i.e. protein hydrolases)
activities;
This is an extremely important
phenomenon: the cell cycle (cell
growth  DNA synthesis  mitosis)
is tightly regulated by the timely
degradation of a series of proteins
called cyclins.
Enzyme
Proteasome
Degraded
enzyme
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Regulation of enzyme activity
5. Limited proteolysis

Several enzymes (particularly digestive
enzymes) are initially synthesized as
inactive precursors (zymogens /
proenzymes);

Activation of the enzymes is done by the
cleavage of a limited number of
peptide bonds (usually 2-3);

The mature enzyme is therefore made
up of 2 to 3 chains, held together by
disulfide bonds
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
5. Limited proteolysis
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
5. Limited proteolysis
Small intestine
Secreted by
pancreas
CHMI 2227 - E.R. Gauthier, Ph.D.
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Regulation of enzyme activity
5. Limited proteolysis

Pancreatic trypsin inhibitor inhibits
trypsin, and prevents trace
amounts of activated trypsin from
triggered the proteolytic cascade
in the pancreas/pancreatic ducts;

Inherited deficiencies in a similar
protease inhibitor (a1-antitrypsin,
which inhibits elastase), leads to
damage to the lungs and
emphysema.

Cigarette smoke also oxidizes a
very important side chain of a1antitrypsin, leading ot its
inactivation and emphysema.
CHMI 2227 - E.R. Gauthier, Ph.D.
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