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


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


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;


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


Regulation of enzyme activity1. Allostery - ATCase


Regulation of enzyme activity1. Allostery - ATCase

ATCase CTP


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


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…);


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;


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


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;


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


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


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


Regulation of enzyme activity5. Limited proteolysis



Small intestine

Secreted bypancreas



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.

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CHMI 2227 - E.R. Gauthier, Ph.D. 1 CHMI 2227E Biochemistry I Enzymes: - Regulation