16 - Regulating Protein Function

Question 1

How is protein (enzyme) activity regulated?

Answer 1

SHORT TERM

• _{Change in substrate or product concentration (fast)}
• _{Change in protein conformation (longer) :}

_{- Proteolytic cleavage (irreversible)}

_{- Covalent Modification (e.g phosphorylation)}

_{- Allosteric regulation}

LONG TERM

• _{Changing rate of protein degradation (ubiquitin-proteasome pathway)}
• _{Changing rate of protein synthesis (enzyme induction)}

Question 2

What are isoenzymes?

Answer 2

Enzymes that catalyse the same reaction but have different kinetic properties, e.g hexokinase and glucokinase

Question 3

What is the difference between hexokinase and glucokinase?

Answer 3

• Glucokinase has a higher Km, so lower affinity, so pathway only activated when blood glucose levels are high

Question 4

What is product inhibition?

Answer 4

Accumulation of a product leads to inhibition of the forward reaction.

e.g glucose-6-phosphate inhibits hexokinase activity

Question 5

What is one of the main differences between a multisubunit (allosteric) enzyme and a single subunit enzyme?

Answer 5

• Allosteric show sigmoidal relationship
• Simple show rectangular parabola

Question 6

Draw a graph to show the relationship between substrate concentration and reaction rate in an allosteric enzyme?

Answer 6

Substrate binding to one subunit causes a conformtational change, making subsequent binding easier

Question 7

What is an allosteric activator and inhibitor?

Answer 7

Bind to enzyme at a site that is not the active site, which induces a conformational change

- Activator: Stabilises R state

- Inhibitor: Stabilises T state, e.g 2,3 BPG, products of pathway

Question 8

Draw a graph of Vi/Vmax against [Substrate], with and without an allosteric activator.

Answer 8

Question 9

What is the role of phospho-fructokinase and what are the allosteric activators and inhibitors?

Answer 9

• Allosterically regulated to set the pace of glycolysis

Activators: AMP, Fructose 2,6-Bisphosphate

Inhibitors: ATP, Citrate, H+

Question 10

Draw a graph of reaction velocity against [phosphofructokinase]

Answer 10

Question 11

What are some covalent modifications that can affect enzyne activity?

Answer 11

• Acetylation
• Carboxylation
• Phosphorylation
• Sulfation
• Ubiquitination

Question 12

What enzymes are involved in phosphorylation/dephosphorylation?

Answer 12

Protein Kinases - Add terminal phosphate from ATP to OH groups on Ser, Thr, Tyr

Protein Phosphatases - Hydrolysis reactions to remove phosphate

Question 13

How does phosphorylating an enzyme affect its activity?

Answer 13

• Adds 2 negative charges
• Phosphate can form H bonds
• Conformational change, especially if near the active site
• Links energy status to metabolism, using ATP
• Allows for amplification affects, e.g PKA

Question 14

What is reciprocal regulation?

Answer 14

e.g Glycogenolysis and Glycogenesis

When one path way is active, the other is inhibited

Question 15

Explain how proteolytic cleavage can affect enzyme activity?

Answer 15

• Digestive enzymes synthesised as zymogens (inactive as active site blocked)
• Cleavage
• Fully active protein

Question 16

Give some examples of zymogens/

Answer 16

Apoptosis mediated by captases, that were initially procaptases

Question 17

What is the proteolytic cleavage of chymotrypsinogen?

Answer 17

Question 18

How is proteolytic cleavage activated?

Answer 18

Trypsin is a protease and cleaves zymogens

Question 19

If proteolytic cleavage is irreversible, how is the enzyme switched off?

Answer 19

• Degradation
• Irreversible binding of inhibitors to active site, especially active site of trypsin

Question 20

How can trypsin inhibitors lead to disease?

Answer 20

• Deficiency of a₁antitrypsin
• Can’t switch off trypsin and therefore other proteases
• Elastase destroys alveolar walls

Question 21

Why are cascades important?

Answer 21

Allow amplification of regulatory signals

Question 22

What are the key features of regulation?

Answer 22

• Can be irreversible or reversible
• Regulation can occur on a range of time scales, seconds to days

Question 23

Summarise the blood clotting cascade and why the cascade is important?

Answer 23

Cascade important as normally little factors in the blood

Question 24

What are the key features of the clotting cascade?

Answer 24

• Fast responsive pathway
• Initial signal amplified by cascade
• Thrombin produce quickly which activates fibrinogen cleavage

Question 25

What is the structure of prothrombin?

Answer 25

- Kringle: Keep prothrombin inactive - Gla domain: Target prothrombin to where damage has occured (serine protease means active site contains serine)

Question 26

What is a Gla domain?

Answer 26

- G-carboxyglutamate residues - Blood clotting factor precursors (2,7,9,10) get extra carboxyl groups added to glutamates during post-translation modification. Lots of -ve charge - At site of damage lots of Ca²⁺, so negative charge on Gla domain attracted to the site, bringing together blood clotting factors and speeding up cascade

Question 27

What is the structure of fibrinogen?

Answer 27

- 2 sets of tripeptides joined by disulphide bonds at N terminus - Fibrous, three globular domains joined by roads - N terminus (middle), highly negative, stops aggregation of fibrinogen

Question 28

How does a fibrin clot form?

Answer 28

- Thrombin cleaves fibrinopeptides A and B at N-terminus - C terminus of B and G globular subunits interact with newly exposed N-terminus, forming clot - Clot stabilised by crosslinking

Question 29

How are fibrin clots stabilisied?

Answer 29

CROSS LINKING - Formation of amide bonds between side chains of glutamine and lysine - Catalysed by transglutaminase which is activated from protransglutaminase by thrombin

Question 30

What is classic haemophillia caused by?

Answer 30

- Defect in Factor VIII - Factor VIII is a cofactor involved in activating Factor X - Without activation of X, no thrombin, fibrin clot cannot be formed - Treat by giving recombinant factor VIII

Question 31

Give examples of where there is amplification in the blood clotting cascade?

Answer 31

- Positive feedback - Thrombin is feedback activator for clotting factors, by allosteric regulation

Question 32

How do you stop the clotting cascade?

Answer 32

**1. Dilution and removal of clotting factors by blood and liver** **2. Degradation of clotting factors** Protein C degrades Factor Va and VIIIa. Thrombin binds to thrombomodulin (endothelial receotir) causing acitvation of protein C. Defect in protein C leads to thrombotic disease **3. Antithrombin III inhibitors bind to thrombin and stop it working** (AT3 works best when bound to heparin. Doesnt act on thrombomodulin bound thrombin

Question 33

What is fibriniolysis and what is it regulated by?

Answer 33

Break down of blood clot (fibrin), tightly regulated

Question 34

Using basic knowledge of fibrinolysis, how can you treat a DVT?

Answer 34

- Give streptokinase or tPA, will cause plasmin to be formed and therefore will activated fibrinolysis

Question 35

Explain the blood clotting cascade very simply.

Answer 35

1. Inactive zymogens present at very low concentrations 2. Proteolytic activation 3. Amplification of initial singal by cascade 4. Clustering of clotting factors at site of damage 5. Feedback activation by thrombin ensures clotting continues 6. Termination of clotting by one of three mechanisms 7. Fibrinolysis controlled by proteolytic activation

Question 36

What are clotting factors?

Answer 36

Cofactors or proteases needed for activation of the next step in the clotting cascade

Question 37

What needs to occur to fibrinogen to form a blood clot (fibrin)?

Answer 37

Proteolytic activation (cleavage)

Question 38

Why are proteases secreted as zymogens?

Answer 38

Prevents them digesting the stomach and pancreas and causing damage to them

Question 39

How does warfarin work?

Answer 39

- Vitamin K analogue - G-carboxyglutamates cannot be formed - Prevents clotting occurring properly as clotting factors can't come together. - Anti-coagulant