S15 Regulation of Protein Function

Question 1

How are enzymes regulated long term?

Answer 1

1. Change in rate of protein synthesis

2. Change in rate of protein degradation

Question 2

How are enzymes regulated short term?

Answer 2

1. Substrate and product concentration

2. Change in enzyme conformation

Question 3

How can you change the conformation of an enzyme (linked to short term regulation)?

Answer 3

1. Allosteric regulation
2. Covalent modification
3. Proteolytic cleavage

Question 4

What is the blood clotting cascade?

Answer 4

1. Intrinsic pathway (damaged endothelial lining) and extrinsic pathway (trauma)
2. Factor X activated
3. Thrombin is activated (prothrombin —> thrombin)
4. Formation of a fibrin clot (fibrinogen —> fibrin)

Question 5

What does the blood clotting cascade allow for?

Answer 5

Allows the formation of a clot from activation of very small amounts of the initial factor

Question 6

What are the domains in prothrombin? What is their role?

Answer 6

• 2 Kringle domains - help keep prothrombin in the inactive form
• Gla domain - target the protease to the appropriate site for activation

Question 7

How does a fibrin clot form?

Answer 7

1. Thrombin cleaves the fibrinopeptides A and B from the central globular domain of fibrinogen
2. The globular domains at the C-terminal of the beta and gamma chains interact with the exposed sequences at the N-termini of the cleaved B and A chains
3. Forms a fibrin mesh/clot
4. Stabilised by the formation of aside bonds between lysine and glutamine residues (catalysed by (protransglutaminase —>) transgulatminase)

Question 8

Which factor has a defect that causes haemophilia?

Answer 8

Factor VIII (antihaemophilic factor)

Question 9

What type of feedback does thrombin cause?

Answer 9

Positive feedback

*an increase in thrombin leads to an increase in factor XIa, VIIIa, Va and XIIIa

Question 10

How is the clotting process stopped?

Answer 10

1. Localisation of (pro)thrombin
2. Digestion by proteases
3. Specific inhibitors

Question 11

How does localisation of (pro)thrombin stop the clotting process?

Answer 11

Leads to dilution of clotting factors by blood flow and are removed by the liver

Question 12

How does digestion by proteases stop the clotting process?

Answer 12

Degradation by proteases activated by thrombin binding to thrombomodulin receptor (negative feedback)

E.g. protein C degrades factors Va and VIIIa
(Defects in protein C can cause thrombotic disease)

Question 13

How do specific inhibitors stop the clotting process?

Answer 13

An example of an inhibitor is antithrombin III (AT3) which is enhanced by heparin binding

Question 14

How is the blood clot removed?

Answer 14

By fibrinolysis

Plasminogen —> plasmin (activated by streptokinase and t-PA)

Plasmin breaks down fibrin to fibrin fragments

Question 15

What are the 7 key control points in blood clotting?

Answer 15

1. Inactive zymogens present at low concentration
2. Proteolytic activation
3. Amplification of initial signal by cascade mechanism
4. Clustering of clotting factors at site of damage
5. Feedback activation by thrombin ensures continuation of clotting
6. Termination of clotting by multiple mechanisms
7. Clot breakdown controlled by proteolytic activation

Question 16

How does the substrate and product concentration regulate enzyme activity?

Answer 16

• substrate availability will affect the rate of enzyme activity
• isoenzymes are different forms of the same enzyme (so have different kinetic properties/parameters e.g. hexokinase and glucokinase)
• accumulation of product inhibits the forward reaction (e.g. G-6-P inhibits hexokinase activity)

Question 17

How does allosteric regulation regulate enzyme activity?

Answer 17

Shows a sigmoidal relationship with T and R states (T - low affinity, R - high affinity)

Substrate binding to one subunit makes subsequent binding to other subunits easier

Question 18

What are allosteric activators and allosteric inhibitors?

Answer 18

Activators - increase the proportion of enzyme in the R state

Inhibitors - increase the proportion of enzyme in the T state

Question 19

What is an example of allosteric regulation?

Answer 19

Phosphofructokinase (PFK) is allosterically regulated

Activated by AMP, fructose-2,6-bisphosphate (low energy)
Inhibited by ATP, citrate and H+ (high energy)

Question 20

How does covalent modification regulate enzyme activity?

Answer 20

• protein kinases - add phosphate Fromm ATP to OH group of Ser, Thr, Tyr
• protein phosphatases - reverse the effects of kinases by hydrologic removal

Question 21

Why is adding a phosphate effective?

Answer 21

• adds a negative charge
• can make H bonds
• allows for amplification
• rate of phosphorylation/dephosphorylation can be adjusted

Question 22

Describe the process of proteolytic activation of chymotrypsinogen.

Answer 22

Trypsin activates chymotrypsin from chymotrypsinogen

Question 23

What is zymogen activation by proteolytic cleavage?

Answer 23

Enteropeptidases activate trypsin (from trypsinogen)

Question 24

How do you inhibit proteolytic cleavage?

Answer 24

With endogenous inhibitors

E.g. pancreatic trypsin inhibitor binds to trypsin and stops the activity

Question 25

How are enzymes activated by proteolytic cleavage?

Answer 25

1. Digestive enzymes are synthesised from zymogens
2. Protein hormones are synthesised as inactive precursor e.g. insulin
3. Blood clotting is mediated by a cascade of proteolytic activations
4. Many developmental processes are controlled by activation of zymogens
5. Apoptosis is mediated by proteolytic enzymes (capsases)