Week 3- Regulation of Protein Activity

Question 1

Long-term regulation of protein activity

Answer 1

Change in rate of protein synthesis: enzyme induction/repression
Change in rate of protein degradation: proteasome-ubiquitin pathway

Question 2

Isoenzymes

Answer 2

Enzymes that catalyse the same reaction but have a different amino acid sequence. They may also have different kinetic properties.

Question 3

Product inhibition of enzymes

Answer 3

Accumulation of the product of a reaction inhibits forward reaction.

For example: glucose-6-phosphate inhibits hexokinase activity

Question 4

Allosteric regulation of enzymes

Answer 4

• Sigmoid relationship between rate and substrate concentration
• Multi-subunit enzymes
• Can exist in two conformations
• Substrate binding to one subunit makes subsequent binding to other subunits progressively easier

Question 5

Allosteric activators and inhibitors

Answer 5

Activators: increase proportion of enzyme in R state
Inhibitors: Increase proportion of enzyme in T state

Question 6

Allosteric regulation of phosphofructokinase

Answer 6

Fructose-6-phosphate + ATP –> Fructose-1,6-bisphosphate + ADP + H+

-Phosphofructokinase sets the pace for glycolysis.
Activators of phosphofructokinase: AMP, Fructose-2,6-bisphosphate
Inhibitors of phosphofructokinase: ATP (high-energy signal), Citrate, H+

Question 7

Zymogen

Answer 7

The inactive precursor of a proteolytic enzyme

Question 8

Covalent modification of enzymes

Answer 8

1. Phosphorylation

Question 9

Protein kinases

Answer 9

Add phosphate groups to particular amino acid residues that have hydroxyl groups;
Transfer terminal phosphate of ATP to hydroxyl group of Serine, Threonine and Tyrosine;

Question 10

Protein phosphatases

Answer 10

Reverse effects of kinases by catalysing hydrolytic removal of phosphoryl groups from proteins

Question 11

List the digestive enzymes synthesised as zymogens in the stomach and pancreas.

Answer 11

Stomach- Pepsinogen (Pepsin)
Pancreas - Chymotrypsinogen (chymotrypsin), Trypsinogen (trypsin), Procarboxypeptidase (carboxypeptidase), Proelastase (elastase)

Question 12

Caspases

Answer 12

Proteolytic enzymes which are synthesised in inactive form and mediate programmed cell death (apoptosis)

Question 13

Give examples of endogenous inhibitors that regulate protease activity.

Answer 13

1. Pancreatic trypsin inhibitor: binds trypsin and stops activity
2. alpha1-antitrypsin: 53 kDa plasma protein that inhibits a range of proteases

Question 14

Emphysema

Answer 14

• Deficiency of alpha1-antitrypsin

- Destruction of alveolar walls by elastase

Question 15

Short-term regulation of enzyme activity

Answer 15

1. Substrate and product concentration

2. Change in enzyme conformation: allosteric regulation, covalent modification, proteolytic cleavage

Question 16

Extrinsic pathway of blood clotting

Answer 16

1. Activation of factor VII: membrane damage exposes extracellular domain of tissue
2. Autocatalytic activation of factor III

Question 17

Intrinsic pathway of blood clotting

Answer 17

1. Membrane damage plays a role in activation of intrinsic pathway
2. Factor IX and X are targeted to membrane by gla domains
3. Ca2+
4. Required for sustained thrombosis activation

Question 18

Activation of thrombin

Answer 18

1. Proteolytic cleavage at Arg 274 to release a fragment containing first three domains
2. Cleavage after Arg 323 releases fully active thrombin consisting of two chains, 6 kDa and 31 kDa linked by disulphide bonds.

Question 19

Role of carboxyglutamate (Gla) residues:

Answer 19

1. Post-translational modification of factors II, VII, IX, X in the liver
2. Addition of COOH groups to glutamate residues to form carboxyglutamate
3. Allows interaction with sites of damage and brings together clotting factors
4. Calcium can act as a cross bridge and attract negatively charged carboxyl groups on clotting factors
5. Process: vitamin K dependent

Question 20

Calcium binding region of prothrombin

Answer 20

• Prothrombin binds calcium via Gla residues
• Only prothrombin next to site of damage will be activated
• clots localised to site of damage

Question 21

Structure of fibrinogen

Answer 21

1. 340 kDa protein
2. 2 sets of tripeptides (alpha, beta and gamma) joined at N-termini by disulphide bonds
3. 3 globular domains linked by rods
4. N-terminal regions of alpha and beta chains are highly negatively charged: prevent aggregations of fibrinogen

Question 22

Formation of fibrin clot

Answer 22

1. Thrombin cleaves fibrinopeptides A and B from central globular domain of fibrinogen
2. Globular domains at C-terminal ends of B and Y chains interact with exposed sequences at N-termini of cleaved beta and alpha chains to form fibrin mesh
3. The newly formed clot is stabilised by the formation of amide bonds between side chains of lysine and glutamine residues in different monomers. This cross-linking reaction is catalysed by transglutaminase, which is activated from protransglutaminase by thrombin.

Question 23

Classic hemophilia

Answer 23

1. Defect in factor VIII
2. Factor VIII stimulates activity of factor IXa (a serine protease)
3. Activity of factor VIII markedly increased by limited proteolysis by thrombin and factor Xa. This positive feedback amplifies clotting signal and accelerates clot formation.
4. Treatment with recombinant factor VIII

Question 24

Ways to stop the clotting process

Answer 24

1. Dilution of clotting factors by blood flow and removal by liver
2. Digestion by proteases: for example, factor Va and factor VIIIa are degraded by protein C; protein C is activated by thrombin binding to endothelial receptor, thrombomodulin
   Defects in protein C –> thrombotic disease
3. Specific inhibitors: antithrombin III binds strongly to factor X: enhanced by heparin binding, AT3 does not act on thrombomodulin bound thrombin

Question 25

Fibrinolysis

Answer 25

Plasminogen —-> Plasmin: Fibrin —> fibrin fragments;

Bacterial enzyme streptokinase/t-PA: activator for plasminogen

Question 26

Key points for blood clotting

Answer 26

1. Feedback activation by thrombin ensures continuation of clotting
2. Clotting factors: proteases/cofactors needed for activation of next step
3. proteolytic cleavage of fibrinogen –> fibrin which aggregates to form clots