CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227F Biochimie I Enzymes: - Concepts de base.
CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Enzymes: - Regulation.
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Transcript of CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Enzymes: - Regulation.
CHMI 2227 - E.R. Gauthier, Ph.D. 1
CHMI 2227EBiochemistry I
Enzymes:- Regulation
CHMI 2227 - E.R. Gauthier, Ph.D. 2
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:
3- Covalent modification: Phosphorylation on Ser/Thr/Tyr
4- Degradation of the enzyme
5- Limited proteolysis
1-Allostery Inhibition by product Activation by
substrate/cofactor
2- Binding of regulatory subunits
CHMI 2227 - E.R. Gauthier, Ph.D. 3
Regulation of enzyme activity1. 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. 4
Regulation of enzyme activity1. 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
AspCarbomoyl phosphate
CHMI 2227 - E.R. Gauthier, Ph.D. 5
Regulation of enzyme activity1. Allostery - ATCase
CHMI 2227 - E.R. Gauthier, Ph.D. 6
Regulation of enzyme activity1. Allostery - ATCase
ATCase CTP
CHMI 2227 - E.R. Gauthier, Ph.D. 7
Regulation of enzyme activity2. 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
Ade
nyla
teC
ycla
se
cAM
PP
hosph
odiesteraseAMP
Caffeine
CHMI 2227 - E.R. Gauthier, Ph.D. 8
Regulation of enzyme activity3. 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. 9
Regulation of enzyme activity3. 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. 10
Regulation of enzyme activity3. Phosphorylation Adrenaline
Adrenaline
G Protein
Adrenaline receptor Adenylate cyclase
ATPcAMP
Protein Kinase A(inactive)
↑Intracellular glucose PKA-cAMP(active)
↑Energy
Run Like Hell!
Glycogen (glucose stores)
Phosphorylase kinase
Phosphorylase Kinase-PO4
GlycogenPhosphorylase-PO4
GlycogenPhosphorylase
Inside the cell
Outside the cell
CHMI 2227 - E.R. Gauthier, Ph.D. 11
Regulation of enzyme activity4. 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. 12
Regulation of enzyme activity4. Regulation of enzyme stability
Ubiquitin: 76 amino acid protein Tags other proteins for
degradation;
Ubiquitin is attached to other proteins by a series of 3 enzymes (E1, E2 and E3);
EnzymeUb
UbUb
Ub
Enzyme
Ub
E1
E2
E3
E1
E2Ub
Ub
E3Ub
CHMI 2227 - E.R. Gauthier, Ph.D. 13
Regulation of enzyme activity4. Regulation of enzyme stability
Polyubiquitylated proteins are targeted to a HUGE protein complex called the proteasome: Contains several subunits with
protease (i.e. protein hydrolases) activities;
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!);
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.
EnzymeUb
UbUb
Ub
Ub
Ub
Ub
Ub
Degraded enzyme
Proteasome
CHMI 2227 - E.R. Gauthier, Ph.D. 14
Regulation of enzyme activity5. 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. 15
Regulation of enzyme activity5. Limited proteolysis
CHMI 2227 - E.R. Gauthier, Ph.D. 16
Regulation of enzyme activity5. Limited proteolysis
Small intestine
Secreted bypancreas
CHMI 2227 - E.R. Gauthier, Ph.D. 17
Regulation of enzyme activity5. 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 (-antitrypsin, which inhibits elastase), leads to damage to the lungs and emphysema.
Cigarette smoke also oxidizes a very important side chain of -antitrypsin, leading ot its inactivation and emphysema.