MCBG Session 8 - Protein Function & Regulation

Question 1

Name 4 different protein (such as enzymes) regulation mechanisms.

Answer 1

1) Isoenzymes - different enzymes forms
2) Allosteric regulation - change in enzyme conformation
3) Phosphorylation - reversible covalent modification
4) Proteolytic activation

Question 2

What are isoenzymes and how do they regulate protein function? - give an example

Answer 2

• Enzymes that catalyse the same reaction but have a different AA sequence - therefore have different activity. Made from different genes or different splicing of same gene.
• Activity can be higher or lower, e.g.: glucokinase is an isoenzyme of hexokinase and increases the rate of reaction.

Question 3

How does allosteric regulation of enzymes work?

Answer 3

• Binding to a site other than the active site to cause a conformational change in the protein.
• Can change the protein into a low affinity state (T-state) which lowers the rate of reaction (done by allosteric inhibitors) or into a high affinity state (R-state) which increases rate of reaction (allosteric activators).
• Does NOT obey Michaelis-Menten kinetics

Question 4

Give an example of an enzyme that undergoes allosteric regulation

Answer 4

PFK-1 - activators that stabilise enzyme into R-state = AMP, Fructose-2,6-BP. Inhibitors that stabilise into T-state = citrate, AMP and H+

Question 5

What is phosphorylation? How can it be reversed?

Answer 5

• Phosphorylation is the transfer of terminal phosphate on ATP to the -OH group of Ser, Thr & Tyr via protein kinases.
• Can be reversed by hydrolytic removal of phosphoryl by protein phosphotases.

Question 6

Why is phosphorylation such an effective regulatory mechanism?

Answer 6

Free energy of PP is large as it adds 2 negative charges and the phosphoryl group can be H-bonds. Also allows for massive amplification effect.
- e.g.: when enzymes activate other enzymes the number of molecules affected increases geometrically in a cascade (can easily get 1,000,000 fold amplification)

Question 7

What is proteolytic activation - when is it important?

Give an example.

Answer 7

Inactive precursor molecules, e.g.: zymogens or proenzymes having peptide bond cleaved (irreversibly) to make precursor active. Important when processes need to be tightly controlled, e.g.: blood clotting, digestive enzymes, apoptosis.
- Enteropeptidases - e.g.: breaking down trypsinogen into active trypsin for digestion in GIT.

Question 8

What are the main differences between Hb and myoglobin?

Answer 8

Hb:

• Carried in blood to transport oxygen all around the body.
• 4 polypeptie chains
• 4 haem groups per molecule

Myoglobin:

• Short term storage of oxygen, supplies it to muscle
• Higher affinity for oxygen
• 1 polypeptide chain + 1 haem group per molecule

Question 9

The oxygen dissociation curve for Hb is sigmoidal - affinity for oxygen increases with PO2 of oxygen - why?

Answer 9

This is due to the co-operativity effect. Once oxygen binds Hb undergoes a conformational change into the R state (allosteric effect), which favours the further binding of oxygen. Therefore co-operativity enhances oxygen transport by Hb. This allows greater oxygen transport compared to myoglobin, as it is more within the R state.

Question 10

Explain the allosteric effects of 2,3 - bisphosphoglycerate (2,3-BPG) on Hb.

Answer 10

• 2,3BPG binds to positively charge residues on B-subunit of Hb.
• This holds subunits together stabilising Hb in the low affinity T-state.
• Therefore favours unloading of oxygen and shifts oxygen dissociation curve to the right.

Question 11

Explain the allosteric effects of H+ and CO2 on Hb. (AKA: the Bohr effect)

Answer 11

• Both H+ and CO2 bind and stabilise Hb in the low-affinity T-state
• This shifts the oxygen dissociation curve to the right
• Allows delivery of oxygen to metabolically active tissues that produce H+ and CO2.

Question 12

Explain the effects of CO on oxygen binding to Hb.

Answer 12

• CO binds to Hb 250 x more readily than O2
• Blocks further oxygen binding once bound
• Stabilised Hb in R state - prevents dissociation at tissues (shifts curve to the left).

Question 13

Describe the molecular basis for sickle cell disease.

Answer 13

1) Mutation of glutamate to valine in B-globin.
2) Val lies on surface in T-state (low affinity)
3) Reduces oxygen carrying capability, cells prone to lyse (anaemia) and are more rigid.
4) This blocks microvasculature such as capillaries.

Question 14

Describe the 2 ways the blood clotting cascade can be activated to form a fibrin clot.

Answer 14

1) Intrinsic (damaged endothelial lining promotes binding of factor Xll) or extrinsic (trauma release tissue factor lll)
2) Both cause factor X activation, leads to thrombin activation
3) Thrombin causes formation of fibrin clot.

Question 15

Why are only very small amounts of initial signal required to trigger formation of a clot?

Answer 15

As amplification occurs during each step in the cascade reaction - as a lot of these reactions are carried out by enzymes such as proteases.

Question 16

Describe the modular structure and cleavage sites of prothrombin.

Answer 16

1) Gla domain at N-terminal - target molecule to appropriate site for activation.
2) 2 x kringle domains keep prothrombin in inactive form
3) Serine protease domain at C-terminal (the thrombin part)
- Cleavage site in-between kringle domain and serine protease.

Question 17

Describe the structure of fibrinogen and how it is converted into fibrin by thrombin.

Answer 17

• 3 polypeptide chains w/2 globular heads separated by rod-like triple-helical alpha helices. Contain fibrinopeptides to prevent fibrinogen molecules coming together and forming a clot (thus keeping it in inactive form)
• Thrombin cuts off fibrinopeptides and fibrin monomers assemble via non-covalent interactions.

Question 18

How is the blood clotting pathway sustained after activation?

Answer 18

1) Factors V and Vlll stimulate activity of other enzymes in pathway
2) Thrombin has positive feedback on factors V, Vlll, Xl & Xlll.

Question 19

What is the role of y-carboxyglutamate residues (Gla)

Answer 19

Adds COOH groups to glutamate residues to form carboxyglutamate (required vit K) - this then becomes a magnetic for clotting factors towards site of damage.

Question 20

What are the 3 ways in which the clotting process can be stopped.

Answer 20

1) dilution of clotting factors by blood flow and removal by liver
2) digestion by proteases
3) Binding of specific inhibitors, e.g.: antithrombin lll

Question 21

How are blood clots broken? (fibrinolysis)

Answer 21

Plasminogen activated by tissue plasminogen activator - produced active plasmin which breaks fibrin down into fibrin fragments.