S10) Regulation of Protein Function

Question 1

How are proteins regulated in the short term?

Answer 1

• Regulate substrate and product concentration
• Change enzyme conformation

Question 2

Identify 3 ways in which enzyme conformation can be altered

Answer 2

• Allosteric regulation
• Covalent modification
• Proteolytic cleavage

Question 3

How can proteins be regulated in the long term?

Answer 3

• Change rate of protein synthesis
• Change rate of protein degradation

Question 4

Explain how protein function can be regulated through [substrate]

Answer 4

• Substrate availability (concentration) affects the rate of enzyme activity
• Some coenzymes will have limited availability e.g. NAD/NADH

Question 5

Explain how protein function can be regulated through [product]

Answer 5

Product inhibition – accumulation of the product of a reaction inhibits the forward reaction e.g. Glucose-6-phosphate inhibits hexokinase activity

Question 6

What are isoenzymes?

Answer 6

Isoenzymes are different forms of the same enzyme that have different kinetic properties

Question 7

What is unique about allosteric enzymes?

Answer 7

Allosteric enzymes show a sigmoid relationship between rate and substrate concentration, instead of the rectangular hyperbola seen for simple enzymes

Question 8

Multi subunit enzymes can exist in 2 different conformations.

Identify them

Answer 8

• T state – low affinity
• R state –high affinity

Question 9

State a benefit of allosteric enzymes

Answer 9

The substrate binding to one subunit makes subsequent binding to other subunits progressively easier

Question 10

Outline allosteric regulation in terms of the actions of allosteric activators and inhibitors

Answer 10

• Allosteric activators - increase the proportion of enzyme in the R state
• Allosteric inhibitors - increase the proportion of enzyme in the T state

Question 11

Illustrate the relative effects of allosteric regulation

Answer 11

Question 12

As an example, describe the allosteric regulation of phosphofructokinase

Answer 12

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

Question 13

Distinguish between the effects of protein kinases and protein phosphatases

Answer 13

• Protein kinases transfer the terminal phosphate from ATP to -OH group of serine, threonine or tyrosine (activate)
• Protein phosphatases reverse the effects of kinases by catalysing the hydrolytic removal of phosphoryl groups from proteins (inactivate)

Question 14

Define the terms phosphorylation and dephosphorylation

Answer 14

• Phosphorylation is the addition of a phosphate group to proteins to signal their activation
• Dephosphorylation is the removal of a phosphate group from a protein to signal its deactivation

Question 15

Why is protein phosphorylation so effective?

Answer 15

• Adds 2 negative charges
• Phosphoryl group makes H-bonds
• Rate of phosphorylation/dephosphorylation can be adjusted
• Links cell energy status to metabolism through ATP
• Allows for amplification effects

Question 16

Explain the amplification of enzyme cascades by proteolytic cleavage

Answer 16

• When enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade
• Amplification of signals by kinase cascades allows amplification of the initial signal by several orders of magnitude within a few milliseconds

Question 17

Provide an example of amplification of enzyme cascades by proteolytic cleavage

Answer 17

Zymogen activation by proteolytic cleavage

Question 18

Identify 5 processes in the body where proteolytic activation is observed

Answer 18

• Digestive enzymes
• Some protein hormones e.g. insulin
• Blood clotting
• Developmental processes
• Apoptosis

Question 19

What is a zymogen?

Answer 19

Zymogens are the inactive form of digestive enzymes which are released in order to prevent an enzyme from digesting the cells which release them

Question 20

Provide a basic outline of the blood clotting cascade

Answer 20

Question 21

Identify the events which trigger the commencement of blood clotting through the intrinsic and extrinsic pathways

Answer 21

• Intrinsic pathway: damaged endothelial lining of the blood cells promotes the binding of factor XII
• Extrinsic pathway: trauma releases tissue factor (factor III) ​resulting in te utocatalytic activation of Factor VII

Question 22

Outline the purpose of the blood clotting cascade

Answer 22

• Both pathways activate Factor X (common endpoint)
• Thrombin is subsequently activated
• Thereafter, a fibrin clot is formed

Question 23

Describe the modular structure of prothrombin

Answer 23

• The protease function (thrombin part) is contained in the C-terminal
• The two kringle domains help keep prothrombin in the inactive form
• Gla domains target it to appropriate sites for its activation

Question 24

State the role of the Gla domain in blood clotting

Answer 24

The Gla domain allows interaction with sites of damage and brings together clotting factors

Question 25

Describe the calcium-binding region of prothrombin

Answer 25

• Prothrombin binds calcium ions via Gla residues
• Only the prothrombin next to the damage site will be activated
• Clots will be localised to the site of damage

Question 26

How is a fibrin clot formed?

Answer 26

⇒ Thrombin cleaves fibrinopeptides A and B from the central globular domain of fibrinogen

⇒ Globular domains at the C-terminal ends interact with exposed sequences at the N-termini to form a fibrin mesh

Question 27

How is the fibrin clot stabilised?

Answer 27

• Amide bonds form 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 28

Identify and describe three means of regulating the blood clotting process

Answer 28

• Localisation of (pro)thrombin – dilution of clotting factors by blood flow, and removal by liver
• Digestion by proteases e.g. factors Va, VIIIa are degraded by protein C
• Specific inhibitors e.g. antithrombin III, enhanced by heparin binding

Question 29

What is classic haemophilia?

Answer 29

• Classic haemophilia, is a genetic disorder caused by missing or defective factor VIII which prevents the stimulated activity of factor IXa, a serine protease
• Treatment with recombinant factor VIII

Question 30

State the 7 key control points in blood clotting (formation & regulation)

Answer 30

​- Low [inactive zymogens]

• Proteolytic activation
• Amplification by cascade mechanism
• Clustering of clotting factors at damage site
• Feedback activation by thrombin ensures continuation of clotting
• Termination of clotting by multiple mechanisms
• Clot breakdown controlled by proteolytic activation