Topic 8: Protein Function And Regulation

Question 1

List the major regulatory mechanisms that control enzyme activity.

Answer 1

• Isoenzymes
• Allosteric regulation
• Phosphorylation
• Proteolytic activation
• Gene expression

Question 2

What are some examples of isoenzymes?

Answer 2

hexokinase and glucokinase - they catalyze the same reaction but have diff amino acid sequence so they have different activity and regulatory properties

Synthesised from different genes or differentially sliced from same gene

Question 3

What is an example of allosteric regulation?

Answer 3

AMP is an allosteic regulator of phosphofructokinase-1, it activates it

Question 4

What is an example of phosphorylation?

Answer 4

Protein kinases add the terminal phosphate from ATP onto the OH group of amino acids

Question 5

What is an example of proteolytic activation?

Answer 5

Controls Blood clotting, digestive enzymes, apoptosis

Question 6

What is allosteric regulation?

Answer 6

Binds to enzyme away from active site which causes conformational changes

Question 7

What are R and T states?

Answer 7

R state = high affinity of enzyme
T state = low affinity of enzyme

States that allosterically regulated enzyme

Question 8

What are allosteric effectors?

Answer 8

Activators - increase proportion of enzyme in R state

Inhibitors - increase proportion of enzyme in T state

Question 9

Give an example of how allosteric regulation works.

Answer 9

Phosphofructokinase-1

Activators: AMP, fructose 2,6-bisphosphate
Inhibitors: citrate, ATP, H+

Question 10

How do enzyme cascades work?

Answer 10

When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade.

Question 11

What is a kinase and phosphatase?

Answer 11

Kinase: enzyme that adds a terminal phosphate from ATP to proteins

Phosphatase: enzyme that catalyze the hydrolysis removal of phosphoryl groups from proteins

Question 12

Why does phosphorylation have an effect?

Answer 12

The free energy of phosphorylation is so large that it allows protein to undergo conformational change.
Adds 2 negative charges and H-bonds which allows diff interactions
Allow for amplification effects

Question 13

What is the definition of the term zymogen?

Answer 13

Inactive precursor molecules

Question 14

How does activation of the clotting cascade lead to formation of fibrin?

Answer 14

Cascade activated intrinsically or extrinsically, which then activates Factor X, which activates thrombin, and that converts fibrinogen into fibrin

Question 15

What are the mechanisms involved in the regulation of clot formation and breakdown?

Answer 15

1. Inactive zymogen 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 localization of prothrombin, dishes Tiong by proteases and binding of specific inhibitors
7. Clot breakdown controlled by proteolytic activation

Question 16

What is the physiological role of myoglobin?

Answer 16

Short term storage of O2

Question 17

What is the physiological role of haemoglobin?

Answer 17

Transport of oxygen around body in blood

Question 18

How does oxygen bind to haem groups?

Answer 18

Oxygen binding changes the position of the iron ion

Question 19

What is the structure of myoglobin?

Answer 19

Only has 1 polypeptide chain so 1 haem group per molecule

Question 20

What is the structure of haemoglobin?

Answer 20

4 polypeptide chains so 4 haem groups

Question 21

Why is the oxygen binding curve for myoglobin hyperbolic?

Answer 21

• Affinity for oxygen is constant

- Each myoglobin only contains one haem group per molecule

Question 22

Why is the oxygen binding curve for haemoglobin sigmoidal?

Answer 22

• Haemoglobin has 4 subunits, so its binding has 4 different equilibrium constants which are interdependent
• One oxygen’s binding influences another oxygen’s binding = homotrophic effect
• Due to this cooperative equilibrium binding = sigmoidal binding curve
• Affinity for oxygen increases w partial pressure of oxygen

Question 23

Why is haemoglobin most suited to its role as an oxygen transporter?

Answer 23

• More haem groups, carry more oxygen
• Sigmoidal properties allows greater oxygen transport compared to comparable proteins entirely in high affinity R state or low affinity T state

Question 24

What are the major structural differences between oxygenated and deoxygenated haemoglobin?

Answer 24

Deoxygenated state = T state = lower affinity

Oxygenated state = R state = higher affinity

Question 25

What are the effects of CO2 and H+ on binding of oxygen by haemoglobin?

Answer 25

• Bohr effect: increase in CO2 or decrease in pH lowers affinity of Hb for oxygen - it stabilizes the T state - moves graph towards right
• This allows delivery of oxygen to metabolically active tissues that produce CO2 and H+

Question 26

What is the effect of 2,3-bisphosphoglycerate on binding of oxygen by haemoglobin?

Answer 26

BPG interacts with positiveLy charged residues on each subunit (in the middle of the quaternary structure)
- stabilizes T state, lowers affinity of Hb for oxygen, moves graph to right

Question 27

What is the effect of carbon monoxide on the binding of oxygen by haemoglobin?

Answer 27

CO binds to haemoglobin a lot more readily than oxygen so it blocks further oxygen binding, stabilizes R state in unaffected subunits, preventing dissociation at tissues

Question 28

How is sickle cell anaemia caused?

Answer 28

• Mutation of negatively charged glutamate to neutral hydrophobic valine in globin
• Valine lies on the surface in T state (deoxygenated), causing haemoglobin to stick to each other
• cells become sickle shaped
• Making it more prone to lyse = anaemia or makes it more rigid

Question 29

What are the key features needed for protein secretion?

Answer 29

• Signal (address), intrinsic to the protein
• Receptor that recognizes the signal and directs it to correct membrane
• Translocation machinery to transport protein across membrane
• Energy to transfer protein to its new location

Question 30

What are the differences between constitutive and regulated secretion?

Answer 30

Constitutive secretion happens all the time continuously, regulated secretion is controlled and only secreted when needed

Question 31

What is the structure of collagen?

Answer 31

• Basic unit is tropocollagen
• 300nm rod-shaped protein
• 3 polypeptides ~1000aa long
• triple helix right handed
• stabilized by H-bonds between chains

Question 32

How is collagen formed?

Answer 32

1. Synthesis and entry of chain into lumen of rough ER
2. Cleavage of signal peptide
3. Hydroxylation of selected proline and lysine residues using prolyl hydroxylase which needs vitamin C and Fe2+ (increases H-bonding to stabilize triple helix)
4. Addition of N-linked oligosaccharides
5. Addition of galactose to hydroxylysine residues
6. Chain alignment, formation of disulfide bonds
7. Formation of triple helical pro collagen from C to N terminus
8. Completion of O-linked oligo-saccharide chains by addition of glucose
9. Packaged into transport vesicle
10. Exocytosis
11. Removal of N and C-terminal propeptides (from procollagen to tropocollagen using procollagen peptidases)
12. Lateral association of collagen molecules followed by covalent cross-linking to form collagen fibril (lysyl oxidase forms covalent bonds between lysine residues)
13. Aggregation of fibrils to form collagen fibre