16 - Regulating Protein Function Flashcards
How is protein (enzyme) activity regulated?
SHORT TERM
- Change in substrate or product concentration (fast)
- Change in protein conformation (longer) :
- Proteolytic cleavage (irreversible)
- Covalent Modification (e.g phosphorylation)
- Allosteric regulation
LONG TERM
- Changing rate of protein degradation (ubiquitin-proteasome pathway)
- Changing rate of protein synthesis (enzyme induction)
What are isoenzymes?
Enzymes that catalyse the same reaction but have different kinetic properties, e.g hexokinase and glucokinase
What is the difference between hexokinase and glucokinase?
- Glucokinase has a higher Km, so lower affinity, so pathway only activated when blood glucose levels are high
What is product inhibition?
Accumulation of a product leads to inhibition of the forward reaction.
e.g glucose-6-phosphate inhibits hexokinase activity
What is one of the main differences between a multisubunit (allosteric) enzyme and a single subunit enzyme?
- Allosteric show sigmoidal relationship
- Simple show rectangular parabola
Draw a graph to show the relationship between substrate concentration and reaction rate in an allosteric enzyme?
Substrate binding to one subunit causes a conformtational change, making subsequent binding easier
What is an allosteric activator and inhibitor?
Bind to enzyme at a site that is not the active site, which induces a conformational change
- Activator: Stabilises R state
- Inhibitor: Stabilises T state, e.g 2,3 BPG, products of pathway
Draw a graph of Vi/Vmax against [Substrate], with and without an allosteric activator.
What is the role of phospho-fructokinase and what are the allosteric activators and inhibitors?
- Allosterically regulated to set the pace of glycolysis
Activators: AMP, Fructose 2,6-Bisphosphate
Inhibitors: ATP, Citrate, H+
Draw a graph of reaction velocity against [phosphofructokinase]
What are some covalent modifications that can affect enzyne activity?
- Acetylation
- Carboxylation
- Phosphorylation
- Sulfation
- Ubiquitination
What enzymes are involved in phosphorylation/dephosphorylation?
Protein Kinases - Add terminal phosphate from ATP to OH groups on Ser, Thr, Tyr
Protein Phosphatases - Hydrolysis reactions to remove phosphate
How does phosphorylating an enzyme affect its activity?
- Adds 2 negative charges
- Phosphate can form H bonds
- Conformational change, especially if near the active site
- Links energy status to metabolism, using ATP
- Allows for amplification affects, e.g PKA
What is reciprocal regulation?
e.g Glycogenolysis and Glycogenesis
When one path way is active, the other is inhibited
Explain how proteolytic cleavage can affect enzyme activity?
- Digestive enzymes synthesised as zymogens (inactive as active site blocked)
- Cleavage
- Fully active protein
Give some examples of zymogens/
Apoptosis mediated by captases, that were initially procaptases
What is the proteolytic cleavage of chymotrypsinogen?
How is proteolytic cleavage activated?
Trypsin is a protease and cleaves zymogens
If proteolytic cleavage is irreversible, how is the enzyme switched off?
- Degradation
- Irreversible binding of inhibitors to active site, especially active site of trypsin
Summarise the blood clotting cascade and why the cascade is important?
Cascade important as normally little factors in the blood
What are the key features of the clotting cascade?
- Fast responsive pathway
- Initial signal amplified by cascade
- Thrombin produce quickly which activates fibrinogen cleavage
What is the structure of prothrombin?
- Kringle: Keep prothrombin inactive
- Gla domain: Target prothrombin to where damage has occured
(serine protease means active site contains serine)
What is a Gla domain?
- G-carboxyglutamate residues
- Blood clotting factor precursors (2,7,9,10) get extra carboxyl groups added to glutamates during post-translation modification. Lots of -ve charge
- At site of damage lots of Ca2+, so negative charge on Gla domain attracted to the site, bringing together blood clotting factors and speeding up cascade
What is the structure of fibrinogen?
- 2 sets of tripeptides joined by disulphide bonds at N terminus
- Fibrous, three globular domains joined by roads
- N terminus (middle), highly negative, stops aggregation of fibrinogen
How does a fibrin clot form?
- Thrombin cleaves fibrinopeptides A and B at N-terminus
- C terminus of B and G globular subunits interact with newly exposed N-terminus, forming clot
- Clot stabilised by crosslinking
How are fibrin clots stabilisied?
CROSS LINKING
- Formation of amide bonds between side chains of glutamine and lysine
- Catalysed by transglutaminase which is activated from protransglutaminase by thrombin
What is classic haemophillia caused by?
- Defect in Factor VIII
- Factor VIII is a cofactor involved in activating Factor X
- Without activation of X, no thrombin, fibrin clot cannot be formed
- Treat by giving recombinant factor VIII
Give examples of where there is amplification in the blood clotting cascade?
- Positive feedback
- Thrombin is feedback activator for clotting factors, by allosteric regulation
How do you stop the clotting cascade?
1. Dilution and removal of clotting factors by blood and liver
2. Degradation of clotting factors
Protein C degrades Factor Va and VIIIa. Thrombin binds to thrombomodulin (endothelial receotir) causing acitvation of protein C. Defect in protein C leads to thrombotic disease
3. Antithrombin III inhibitors bind to thrombin and stop it working
(AT3 works best when bound to heparin. Doesnt act on thrombomodulin bound thrombin
What is fibriniolysis and what is it regulated by?
Break down of blood clot (fibrin), tightly regulated
