Regulation Of Protein Function

Question 1

Name methods of short term regulation

Answer 1

1. Substrate and product concentration
2. Change in enzyme conformation
   A. Allosteric regulation
   B. Covalent modification
   C. Proteolytic cleavage

Question 2

Name methods of long term regulation

Answer 2

LONG TERM REGULATION

1. Change in rate of protein synthesis
2. Change in rate of protein degradation

Question 3

What is the easiest way of controlling enzyme activity?

Answer 3

Changing substrate concentration

Question 4

What are isoenzymes?

Answer 4

Different forms of the same enzyme that have different kinetic properties

Question 5

What is product inhibition?

Answer 5

Accumulation of the product of a relation inhibits the forward reaction

Question 6

Give an example of end product inhibition

Answer 6

Glucose-6-phosphate inhibits hexokinase activity

Question 7

Describe the shape of the rate/substrate conc graph shown by a simple enzyme

Answer 7

Rectangular hyperbola

Question 8

Describe the shape of the rate/substrate conc graph shown by an allosteric enzyme

Answer 8

Sigmoid

Question 9

How many states can multisubunit enzymes exist in? What are these states?

Answer 9

2 conformations
T state - low affinity
R state - high affinity

Question 10

What is positive cooperativity?

Answer 10

Substrate binding to one subunit makes subsequence binding to other subunits progressively easier

Question 11

WHat do allosteric activators do?

Answer 11

Increase the proportion of the enzyme in the R state - rate then increases

Question 12

What do allosteric inhibitors do?

Answer 12

Increase the proportion of the enzyme in the T state - rate then decreases

Question 13

What is the function of phosphpfruktokinase?

Answer 13

Sets the pace of glycolysis and is allosterically regulated

Question 14

Does ATP inhibit or activate PFK? Explain.

Answer 14

ATP in low concs is a substrate
In high concs is an inhibitor which stabilises the T state - as if enough atp is present, glycolysis does not need to take place (as quickly)

Question 15

Does AMP inhibit or activate PFK? Explain.

Answer 15

AMP is a low energy signal
When a cell is very low on ATP it will start converting ADP (ADP+ADP->ATP+AMP)
High levels of AMP means ATP is low
Therefore AMP stabilises R state

Question 16

Does citrate inhibit or activate PFK? Explain.

Answer 16

Citrate is a high energy signal
It is the first product of the Krebs cycle. If it builds up this is a signal that glycolysis can slow down
Therefore stabilises t state - inhibitor

Question 17

Does H+ inhibit or activate PFK? Explain.

Answer 17

H+ is a product - high concentrations is a high energy signal so high concs stabilise the t state

Question 18

Is fructose-2,6-bisphosphate an activator or inhibitor of PFK?

Answer 18

PFK 2 makes this
But it binds to PFK 1 and stabilises the R state
Activator

Question 19

What is the function of protein kinases?

Answer 19

Transfer the terminal phosphate from ATP to the -OH group of Ser, Thr, Tyr
Covalent addition

Question 20

What is the function of protein phosphatases?

Answer 20

Reverse the effects of protein kinases by catalysing the hydrolysis removal of phosphoryl groups from proteins
This can increase of decrease activity

Question 21

Why is protein phosphorylation so effective?

Answer 21

Adds 2 negative charges
A phosphoryl group can make H-bonds
Rate of phosphorylation/dephosphorylation can be adjusted
Links energy status of the cell to metabolism through ATP
Allow for amplification effects
How does this have an affect of a protein?
-ve charge - big effect on protein structure
Allows protein to make additional hydrogen bonds
We can regulate rate of phosphorylation/dephosphoylation

Question 22

Describe amplification by enzyme cascades

Answer 22

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 23

Name some enzymes which are activated by specific proteolytic cleavage

Answer 23

Specific proteolysis is a common means of activating enzymes in biological systems. For example:
1. Digestive enzymes are synthesized as zymogens (inactive precursors) in the stomach and pancreas.
2. Some protein hormones (e.g. insulin) are synthesised as inactive
precursors.
3. Blood clotting is mediated by a cascade of proteolytic activations that ensures a rapid and amplified response.
4. Many developmental processes are controlled by the activation of zymogens to contribute to tissue remodelling.
5. Programmed cell death (apoptosis) is mediated by proteolytic
enzymes, caspases, which are synthesised in inactive (procaspase) form.

Question 24

What are zymogens?

Answer 24

Inactive precursors of enzymes

Question 25

Give some examples of zymogens

Answer 25

Pepsinogen - stomach 
Chymotrypsinogen - pancreas
Trypsinogen - pancreas
Proelastase - pancreas
Procarboxypeptidase - pancreas

Question 26

What is the function of endogenous inhibitors?

Answer 26

Regulate protease activity e.g.pancreatic trypsin inhibitor binds trypsin ans stops activity

Question 27

What causes emphysema?

Answer 27

Deficiency of alpha1-antitrypsin, destruction of alveolar walls by elastase

alpha1-antitrypsin - plasma protein that inhibits a range of proteases

Question 28

What are the 2 pathways to start the blood clotting cascade?

Answer 28

Intrinsic pathway - damaged endothelial lining of blood cells promotes binding of factor XII
Membrane damage plays a role in activation of the intrinsic pathway
• Factor IX and X are targeted to membrane by Gla domains
• Ca2+ plays a role
• required for sustained thrombin activation

Extrinsic pathway - Trauma releases tissue factor III - Membrane damage exposes extracelluar domain of tissue (factor III) Auotcatalytic activation of Factor VII

Factor X is the common endpoint for both pathways

Question 29

What does factor X activation lead to?

Answer 29

Thrombin activation then formation of fibrin clot

Question 30

Why is a cascade beneficial?

Answer 30

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

Question 31

Describe the modular structure or prothrombin

Answer 31

Gla domain - Kringle - Kringle - - serine protease

Question 32

Where is the protease function of prothrombin?

Answer 32

C-terminal domain

Question 33

What is he function of the Kringle domains?

Answer 33

Help keep prothrombin in the inactive form

Question 34

What is the purpose of the gla domains?

Answer 34

Target prothrombin to appropriate sites for activation

Gla = carboxy glutamate

Question 35

What happens when prothrombin is activated?

Answer 35

Releases active serine protease

Question 36

Describe the role of gla domains

Answer 36

Post-translational modification of factors II, VII, IX, X in the liver
• Addition of COOH groups to glutamate residues to form carboxyglutamate (Gla)
• Allows interaction with sites of damage and brings together clotting factors

In a damages blood vessels there are exposed phospholipids - Ca2+ binds to site of damage
Gla residues have COO-so are -ve charged so attracted by Ca2+
Clotting factors contain gla domains so are attracted to site of damage - if theyre closer together they work faster
Only prothrombin next to site of damage will be activated so clots will be localised to the site of damage

Question 37

Describe the structure of fibrinogen

Answer 37

340kDa protein
• 2 sets of tripeptides , a, b, g, joined at N-termini by disulphide bonds
• 3 globular domains linked by rods
N-terminal regions of a and b chains are highly negatively charged and prevent aggregation of fibrinogen

Question 38

Describe the formation of a fibrin clot

Answer 38

(1) Thrombin cleaves fibrinopeptides A and B from the central globular
domain of fibrinogen.
(2) Globular domains at the C-terminal ends of the b and g chains interact with exposed sequences at the N-termini of the cleaved b and a chains to form a fibrin mesh or clot.
The newly formed clot is stabilised by the formation of amide bonds 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 39

What is haemophilia

Answer 39

Defect in factor VIII
- Factor VIII (‘antihaemophilic factor’) is not a protease, but markedly
stimulates the activity of factor IX, a serine protease.
- The activity of factor VIII is markedly increased by limited proteolysis by thrombin and factor Xa. This positive feedback amplifies the clotting signal and accelerates clot formation.
- Treatment with recombinant factor VIII

Question 40

How is the clotting process stopped?

Answer 40

1. Localisation of (pro)thrombin - Dilution of clotting factors by blood flow, and removal by liver
2. Digestion by proteases - for example, factors Va and VIIIa are degraded by protein C
   - protein C is activated by thrombin binding to endothelial receptor, thrombomodulin
   - defects in protein C can cause thrombotic disease

Question 41

How is the clotting process regulated?

Answer 41

Inhibitors stop clotting progress
Heparin binds to form tight complex - blocks thrombin activity

3. Specific inhibitors
Antithrombin III (AT3)
Enhanced by heparin binding
AT3-heparin does not act on
thrombomodulin-bound thrombin

Question 42

Give an overview of the key points in blood clotting

Answer 42

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.