Enzymes and Blood Clotting Cascade

Question 1

How do enzymes work?

Answer 1

They lower activation energy of the reaction. Binding of substrate to active site increases local concentration of reactant and also stabilises formation of high energy transition state

Question 2

What does Vmax signify?

Answer 2

Maximum velocity when enzyme saturated with substrate.

Question 3

What does Km signify?

Answer 3

Km is substrate concentration that gives half of Vmax. The lower the Km, the higher the affinity for substrate.

Question 4

Describe an example of an allosterically regulated enzyme

Answer 4

Phosphofructokinase. It sets the pace of glycolysis. It is activated by AMP, fructose-2-6-bisphosphate. It is inhibited by ATP, citrate, H+

Question 5

What are zymogens?

Answer 5

Inactive enzyme precursors that become activated by cleavage with another enzyme.

Question 6

Describe some ways in which enzymes are regulated

Answer 6

Allosteric regulation, Positive co-operativity (e.g haemoglobin), covalent modification, proteolytic activation

Question 7

Describe the action of kinase enzymes

Answer 7

Transfer phosphate groups to -OH group of amino acid from ATP. (Serine, Threonine, Tyrosine)

Question 8

Describe the action of phosphotase enzymes

Answer 8

Remove phosphate groups through hydrolytic activity. Phosphate groups are bulky, charged groups, so they affect enzyme conformation.

Question 9

Describe the role of the intrinsic and extrinsic pathways in the blood clotting cascade. Also, what is the role of positive feedback in the cascade?

Answer 9

Extrinsic pathway is activated by membrane damage (factor 3 involved).

Intrinsic pathway activated by external trauma, such as blunt force (factor 12involved).

Factor X activated by both

Positive feedback in the way that activation of thrombin promotes further activation in cascade.

Question 10

How is the blood clotting cascade regulated?

Answer 10

Zymogen concentration; digestion by proteases; specific inhibitors (e.g Heparin binding to antithrombin-3, AT3-Heparin doesn’t act on thrombulin-bound thrombin).

Question 11

Describe fibrinolysis

Answer 11

Fibruinolysis is breaking down of clot. T-PA and streptokinase promote plasminogen becoming plasmin. Plasmin promotes fibrin breaking into fibrin fragments.

T-PA is tissue plasminogen activator

Question 12

What are the differences between irreversible and reversible enzyme inhibitors?

Answer 12

Irreversible inhibitors bind very tightly and form covalent bonds, i.e. sarin

Reversible inhibitors can freely dissociate

Question 13

How do competitive and non-competitive enzymes affect Km and Vmax?

Answer 13

Competitive- binds at active site, affects Km (increases it) not Vmax. adding enough substrate will always overcome substrate

Non-competitive- binds at another site on the enzyme. affects Vmax (lowers it) but not Km

Question 14

Give an example of product inhibition in the body

Answer 14

Hexokinase is inhibited by glucose-6-phosphate

Question 15

Why is phosphorylation so effective

Answer 15

• links energy status of cell to metabolism (ATP)

- allows for amplification effects

Question 16

Why are digestive enzymes synthesised as zymogens?

Answer 16

So they don’t digest the cell itself

Question 17

What does trypsin do?

Answer 17

• activates all other digestive enzymes
• trypsinogen is the inactive form
• enteropeptidase catalyses the conversion
• pancreatic trypsin inhibitor binds trypsin and stops activity

Question 18

What is the clinical significance of alpha-1 antitrypsin deficiency?

Answer 18

emphysema

Question 19

What are gla domains?

Answer 19

• vitamin K dependent (in the liver, modifies factors)
• calcium required
• targets factors to membrane

Question 20

How is a fibrin clot formed?

Answer 20

1) Thrombin cleaves fibrinopeptides A and B from the central globular domain of fibrinogen.
2) Globular domains at the C-terminal ends of the b and g chains interact with exposed sequences at the N-termini of the cleaved b and a chains to form a fibrin mesh or clot.

The newly formed clot is stabilised by the formation of amide bonds between the 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 21

Describe the condition of haemophilia

Answer 21

Factor VIII (‘antihaemophilic factor’) is not a protease, but markedly stimulates the activity of factor IXa, which is a serine protease. Factor IXa catalyses the formation of factor Xa.

Haemophilia is a defect in this factor.

The activity of factor VIII is markedly increased by limited
proteolysis by thrombin and factor Xa. This positive feedback amplifies the clotting signal and accelerates clot formation.

When factor VIII isn’t present, this doesn’t occur and excessive bleeding results. Treatment with recombinant factor 8.

Question 22

How is the clotting process stopped? (3 ways)

Answer 22

• localisation of prothrombin. Dilution of clotting factors of blood flow, and removal by liver
• digestion by proteases (e.g protein C degrades factor v and VIIa. It is activated by thrombin binding to endothelial receptor, thrombomodullin)
• specific inhibitors (e.g AT3, which is enhanced by heparin bind. AT3-heparin does not act on thrombomodulin-bound thrombin however)