Regulation Of Protein Function

Question 1

Name 10 ways in which protein activity can be regulated

Answer 1

-Using extracellular signals
-Transcription of specific gene(s)
-mRNA degradation
-mRNA translation on ribosome
-Protein degradation
-Enzyme sequestered in subcellular organelle eg ER
-Enzyme binds to substrate
-Enzyme binds to a ligand (allosteric)
-Enzyme undergoes phosphorylation or dephosphorylation
—Enzyme combines with regulatory proteins

Question 2

Name the ways in which enzyme activity is regulated, on the short term.

Answer 2

• Changes in substrate and product concentration cause almost instantaneous changes.
• Change in Enzyme conformation eg allosteric regulation, covalent regulation, proteolytic cleavage.

Question 3

Name the ways in which enzyme activity is regulated, on the long term. And give examples.

Answer 3

• Change in rate of protein synthesis.

- Change in rate of protein degradation.

Question 4

What are isoenzymes?

Answer 4

Isoenzymes are different forms of the same enzyme that have different kinetic properties. These are usually made from the same genes but are alternatively spliced. Eg Hexokinase and Glucokinase

Question 5

What are the differences between Hexokinase and Glucokinase?

Answer 5

Hexokinase has a much lower Km so it has a much higher affinity for glucose and reaches capacity very quickly. Glucokinase has a much higher Km. This means that it is only active when the concentration of glucose is high in the liver (after eating) to quickly lower the amount of glucose. This is a slower enzyme substrate reaction as it takes much lower to reach near Vmax.

Question 6

What is product inhibition?

Answer 6

Accumulation of the product of a reaction inhibits the forward reaction. Eg Glucose-6-phosphate inhibits Hexokinase activity.

Question 7

What shape is the curve for 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

Why do allosteric show this shaped curve?

Answer 8

Allosteric show a sigmoid relationship because they are multisubunit enzymes that can exist in two different conformations (T state - low affinity and R state - high affinity). Substrate binding to one subunit makes subsequent binding to other subunits progressively easier.

Question 9

What is the role of allosteric activators?

Answer 9

Allosteric activators increase the proportion of enzymes in the R state. They bind outside of the active site of the allosteric enzyme to promote the R state.

Question 10

What is the role of allosteric inhibitors?

Answer 10

They increase the proportion of enzymes in the T state.

Question 11

What is phosphofructokinase? How is it allosterically regulated?

Answer 11

Phosphofructokinase (pfk) is the rate limiting step of glycolysis. It is allosterically regulated.
Activator : AMP, fructose-2,6-bisphosphate
Inhibitors: ATP, citrate, H+

Question 12

What are the role of protein kinases?

Answer 12

Protein kinases phosphorylate enzymes by transferring the terminal phosphate from ATP to the -OH group of Ser, Thr, Tyr. Phosphorylation changes (increase or decrease) the activity of enzymes. Eg Glycogen phosphorylase which is involved in glucose homeostasis and energy transduction.

Question 13

What enzyme reverses the effect of protein kinases?

Answer 13

Protein phosphatases. These reverse the effect of protein kinases by catalysing the hydrolytic removal of phosphoryl groups from proteins.

Question 14

Why is protein phosphorylation so effective?

Answer 14

• It adds two negative charges (causes a change in conformation)
• A phosphoryl (PO3 2-) group can make hydrogen bonds
• Rate of phosphorylation / dephosphorylation can be adjusted (activity of kinases and phosphatases)
• Links energy status of the cell to metabolism through ATP
• Allows for amplification effects (cascade).

Question 15

How are the effects of enzymes amplified?

Answer 15

The effect of enzymes can be amplified by the effect of enzyme cascades.
When one enzyme activates other enzymes, the number of affected molecules increases geometrically in an enzyme cascade.
The amplification of signals by kinase cascades allows amplification of the initial signal by several orders of magnitude within a few seconds.

Question 16

Why and how is Glycogen reciprocally regulated?

Answer 16

Glycogen breakdown and synthesis are reciprocally regulated so that only one can be activated at once. The activation of Glycogen phosphorylase switched off Glycogen synthase

Question 17

Give examples of enzymes that are activated by specific proteolytic cleavage (proteolysis)

Answer 17

• Digestive enzymes synthesised as Zymogens in the stomach and pancreas
• So protein hormones (eg insulin) are synthesised as Zymogens
• Blood clotting is mediated by a cascade of proteolytic activations that ensure a rapid and amplified response
• Many developmental processes are controlled by the activation of Zymogens to contribute to tissue remodelling
• Apoptosis is mediated by proteolytic enzymes, caspases, which are synthesised in inactive (procaspase) form.

Question 18

What are Zymogens? Why are they useful?

Answer 18

Zymogens are inactive precursors. It is important that enzymes are made as zymogens so that they don’t break down the cells they are made in.

Question 19

Describe the role of trypsin.

Answer 19

Trypsin is an enzyme (protease) that activates chymotripsinogen. It cleaves it between amino acids 15-16 which then cause it to self activates by further cleaving itself into three amino acid chains.
Trypsin itself is activated by Enteropeptidase and it activates other pancreatic proteases too.
It causes a cascade effect.

Question 20

How can trypsin activity be stopped?

Answer 20

Pancreatic trypsin inhibitor binds to trypsin very tightly and therefore stops its activity (it cannot become trypsinogen because it underwent a permanent change). Alpha 1 antitrypsin is an 53kDa plasma protein that inhibits a range of proteases.

Question 21

What disease can be caused if alpha1-antitrypsin is deficient?

Answer 21

Emphysema.
This is because trypsin catalyses the formation of elastase from proelastase and if this is uncontrolled elastase can cause the destruction of alveolar cell walls.

Question 22

What are the two pathway that can activate the blood clotting cascade?

Answer 22

• Intrinsic pathway. This is triggered by damage to the endothelial lining of blood cells it promotes the binding of factor VII.
• Extrinsic pathway. This is caused by trauma and releases tissue factor III.

Question 23

What is the common end point for both pathways? (Last 3 main steps)

Answer 23

Both pathways activate factor X which activates thrombin and causes the formation of a fibrin clot.

Question 24

How would the blood clotting mechanism be regulated?

Answer 24

As a CASCADE effect because, in the pathways all the factors (mostly proteins) need to come together and cause a clot. To do this, one factor activates the next one in a cascade like effect.
PROTEOLYTIC regulation because some of the proteins need to be cleaved to become activated.
ALLOSTERIC regulation because thrombin allosterically activates some of the other steps. So, as soon as thrombin is activated, some of the other steps become self sustaining.

Question 25

What is a gla domain?

Answer 25

Gla (carboxyglutaate domains) domains target it to appropriate sites for its activation.
It allows for the post translational modification of factors II, VII, IX, X in the liver.
It allows interaction with sites of damage and brings together clotting factors. This happens because calcium (positive) is at site of damage and all clotting factors with gla domains (negative) are attracted to the calcium.
It allows for the addition of extra carboxyl residues (catalysed by vitamin K)

Question 26

Describe the structure of prothrombin and how it becomes thrombin.

Answer 26

Porthrombin is made from a gla domain then two Kringle domains then the cleavage site and the serine protease. This serine protease is the thrombin part of the molecule so, it is cleaved from the rest to become thrombin.

Question 27

Describe the structure of fibrinogen

Answer 27

Is is made of three large proteins (alpha, beta, gamma) which are joined t the N termini by disulphide bonds.
It is made of three globular domains linked by rods.
N-terminal regions of the alpha and beta chains the highly negatively charged and so prevent aggregation of fibrinogen.

Question 28

How do fibrinogen molecules form a clot?

Answer 28

Thrombin cleaves off fibrinopeptides A and B from the central globular domain of fibrinogen.
Globular domains at the C-terminal ends of the beta and gamma chains interact with exposed sequences at the N termini of the cleaved beta and alpha chains to form a fibrin mesh or clot.
Further cross linking occurs between glutamine and lysine residues by the formation of amide bonds. This is catalysed by transglutaminase.

Question 29

What factor is deficient in classic haemophilia?

Answer 29

Factor VIII. This is involved in the activation of factor 10 so is important.

Question 30

How is the clotting process stopped?

Answer 30

• Clotting factors can be diluted by blood flow and removal by the liver.
• Digestion by proteases eg factor V and factor VIIIa are degraded by protein C. This protein is activated by thrombin binding to endothelial receptor, thrombomodulin. If you have defects in protein C, you can get thrombotic disease.
• Specific inhibitors which can bind to thrombin and stop it working eg AT3. This is enhanced by heparin which is. A drug people can take to reduce clotting.

Question 31

How do you get rid of a blood clot ()?

Answer 31

Using fibrinolysis. Plasmin (made as plasminogen which is then activated by factor or Bacteria) breaks fibrin into fragments.