Regulation Of Protein Activity Flashcards
How could you control the regulation of proteins in the long term?
- Change rate of protein synthesis
- Change rate of protein degradation
Define an ISOENZYME and give an example of these
- Different forms of the same enzyme which have different kinetic properties (Km values)
- e.g. Hexokinase (low Km for glucose) and Glucokinase (high Km for glucose, so is only active when conc of glucose in blood in high)
What is meant by product inhibition? Give an example of where this occurs.
- Accumulation of the product of an enzyme controlled reaction inhibits the forward reaction
- e.g. PFK is inhibited by high levels of Fructose-1,6-bisphosphate
What kind of graph would you expect for a protein that is allosterically regulated?
SIGMOIDAL relationship between [S] and rate of reaction v
Explain why allosterically regulated proteins show a sigmoidal relationship
- Allosterically regulated protein usually have multiple subunits e.g. Haemoglobin
- Substrate binding to one subunit is difficult at first but promotes the subsequent binding of substrate to other subunits (subsequent binding is progressively easier)
What is the effect of an allosteric activator?
- Increase the proportion of enzymes in the high affinity R state
- Curve shifts to the LEFT
How does an allosteric inhibitor affect the rate of reaction?
- Decreases rate of reaction
- Binds somewhere other than active site and promotes stabilisation of the low affinity T state
- Curve shifts to the RIGHT
Why is Km insignificant when allosteric regulation is involved?
- Km is used for hyperbolic relationships
- Allosteric regulation shows a sigmoidal relationship
Name 2 substances which act as allosteric activators for PFK
- AMP
- Fructose-6-phosphate
Give an example of a covalent modification mechanism for modifying proteins
- POST-TRANSLATIONAL PHOSPHORYLATION
- Proteins phosphorylated using KINASE enzymes
- Proteins dephosphorylated using PHOSPHATASE enzymes
Which 3 amino acid residues can be phosphorylated and why?
- Serine
- Threonine
- Tyrosine
- All contain -OH groups on their side chains which can accept terminal Pi from ATP
What type of bond is formed during phosphorylation?
COVALENT
How does a phosphatase enzyme work?
- DEPHOSPHORYLATION
- Catalyses the hydrolytic removal of phosphoryl groups from proteins
- Uses water to break the covalent bond
Explain what is meant by ‘amplification’ of a signal and how this causes an enzyme cascade
- Binding of receptor (signal) activates an enzyme (induces a kinase to phosphorylate)
- Activated enzyme goes on to further activate other enzymes
- Number of activated enzymes increases geometrically until there are thousands
- One signal can activate thousands of enzymes in a few seconds due to AMPLIFICATION of the signal
Name 3 proteins which are activated by proteolytic cleavage
- Pepsin
- Trypsin
- Insulin
- Thrombin
- Fibrin
What is a zymogen?
Inactive precursor of a protein
What is the significance of the activation of trypsin in the pancreas?
- Enteropeptidase enzyme in pancreas activates trypsin by proteolytic cleavage of trypsinogen
- Trypsin then activates subsequent pancreatic proteases by stimulating the proteolytic cleavage of each
How is protease activity regulated?
- ENDOGENEOUS INHIBITORS
- Once a zymogen is activated it cannot be switched off
- Highly specific inhibitors are released which bind to active enzymes and inhibit their activity
Give examples of reversible and irreversible mechanisms of protein regulation
- REVERSIBLE - phosphorylation
- IRREVERSIBLE - proteolytic cleavage
Name 4 ways in which proteins can be regulated in the short term
- Substrate and product concentrations (feedback inhibition)
- Change in enzyme conformation by allosteric regulation, covalent modification or proteolytic cleavage
What mechanism of damage might activate the intrinsic pathway of the blood clotting cascade?
Damaged endothelial lining of blood vessels
What do both the intrinsic and extrinsic pathways have in common?
Both lead to activation of Factor X
What is an ENDOPEPTIDASE?
Enzyme that breaks peptide bonds in specific places
Explain how clots are localised to the site of damage
- Damage to endothelial phospholipid bilayer attracts positively charged Ca2+ (Factor IV)
- Calcium bridge is formed (highly positive)
- Attracts highly negative Gla domains on Factors II, VII, IX and X tot he site of damage
- Prothrombin (Factor II) binds to Ca2+ and will only be activated at the site if damage
How are Gla domains formed?
- Post translational modification of Glu residues on Factors II, VII, IX and X
- Addition of COOH forms γ-carboxyglutamate which are highly NEGATIVE
- Requires vitamin K
Which factors are activated in the intrinsic pathway?
- Factor XI
- Factor IX
- Activation of co-Factor XIII
- FACTOR X
What factors are activated in the extrinsic pathway?
- Tissue Factor III
- Factor VII
- FACTOR X
Which factor(s) are involved in the activation of Prothrombin->Thrombin?
Factor X and co-Factor V
What is the role of Factor XIII?
- Transglutaminase
- Cross linking of FIBRIN by linkage of Glutamate and Lysine residues
What is cleaved from prothrombin to activate it to thrombin?
- Gla domain
- 2 Kringle domains
How does Tissue Factor II activate Factor VII?
- Membrane damage exposes extracellular domain of Tissue Factor III
- AUTOCATALYTIC activation of Factor VII
How is polymerisation of fibrinogen prevented when inactive?
NEGATIVE N terminal regions cause fibrinogen molecules to REPEL EACHOTHER
Why is the globular domain of fibrinogen highly negative?
α, β and γ domains linked at N termini by DISULPHIDE BONDS
Describe the formation of a soft clot
- Activation of Thrombin via cascade mechanism
- Thrombin cleaves fibrinopeptides A and B from the central globular domain, forming active FIBRIN
- C terminal of the γ and β globular domains (-ve) are attracted to the N termini of the cleaved α and β chains (+ve) forming a fibrin mesh or soft clot
Describe how a fibrin clot is formed from a fibrin mesh (soft clot)
- Crosslinking between N termini of Glutamine and Lysine residues
- Formation of AMIDE bonds by Factor XIII TRANSGLUTAMINASE which is activated by Thrombin
Why is the blood clotting cascade described as an example of positive feedback?
- Activation of Thrombin through the cascade of activation of previous Factors and co-Factors
- Thrombin can go back through the cascade and activate previous clotting Factors XIII, XI, VIII and V
- This increases the number of active clotting Factors which then increases the amount of active Thrombin
What is protein C?
- Protease enzyme involved in stopping the clotting process
- Activated by thrombin binding to specific receptor THROMBOMODULIN
- Degrades active co-Factors Va and VIIIa
What factor is deficient in Haemophilia A (classic)?
Factor VIII
Describe the methods in which the clotting process can be stopped
- Dilution of clotting factors by blood flow and removal by liver
- Digestion of co-Factors V and VIII by protein C
- Inhibition of thrombin by Antithrombin III
Describe the structure of fibrinogen
- CRAB
- 2 sets of tripeptides containing α, β, γ chains
- Linked at N termini by DISULPHIDE BONDS
- 3 globular domains (C termini) linked by rods
How is haemophilia A treated?
Recombinant Factor VIII
What is plasmin and how is it activated?
- Serine ENDOPEPTIDASE that is involved in the degradation and breakdown of fibrin clots
- Inactive zymogen PLASMINOGEN activated by tissue-plasmin activator t-PA
- Also activated by Streptokinase (drug used to breakdown clots in coronary arteries)
Which clotting factors have Gla domains?
- Factor II (Thrombin)
- Factor XI
- Factor VII
- Factor X
What vitamin is essential for clotting?
Vitamin K
