S10) Regulation of Protein Function Flashcards
How are proteins regulated in the short term?
- Regulate substrate and product concentration
- Change enzyme conformation
Identify 3 ways in which enzyme conformation can be altered
- Allosteric regulation
- Covalent modification
- Proteolytic cleavage
How can proteins be regulated in the long term?
- Change rate of protein synthesis
- Change rate of protein degradation
Explain how protein function can be regulated through [substrate]
- Substrate availability (concentration) affects the rate of enzyme activity
- Some coenzymes will have limited availability e.g. NAD/NADH
Explain how protein function can be regulated through [product]
Product inhibition – accumulation of the product of a reaction inhibits the forward reaction e.g. Glucose-6-phosphate inhibits hexokinase activity
What are isoenzymes?
Isoenzymes are different forms of the same enzyme that have different kinetic properties
What is unique about allosteric enzymes?
Allosteric enzymes show a sigmoid relationship between rate and substrate concentration, instead of the rectangular hyperbola seen for simple enzymes
Multi subunit enzymes can exist in 2 different conformations.
Identify them
- T state – low affinity
- R state –high affinity
State a benefit of allosteric enzymes
The substrate binding to one subunit makes subsequent binding to other subunits progressively easier
Outline allosteric regulation in terms of the actions of allosteric activators and inhibitors
- Allosteric activators - increase the proportion of enzyme in the R state
- Allosteric inhibitors - increase the proportion of enzyme in the T state
Illustrate the relative effects of allosteric regulation
As an example, describe the allosteric regulation of phosphofructokinase
- Activators: AMP, fructose-2,6-bisphosphate
- Inhibitors: ATP, citrate, H+
Distinguish between the effects of protein kinases and protein phosphatases
- 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)
Define the terms phosphorylation and dephosphorylation
- 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
Why is protein phosphorylation so effective?
- 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
Explain the amplification of enzyme cascades by proteolytic cleavage
- 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
Provide an example of amplification of enzyme cascades by proteolytic cleavage
Zymogen activation by proteolytic cleavage
Identify 5 processes in the body where proteolytic activation is observed
- Digestive enzymes
- Some protein hormones e.g. insulin
- Blood clotting
- Developmental processes
- Apoptosis
What is a zymogen?
Zymogens are the inactive form of digestive enzymes which are released in order to prevent an enzyme from digesting the cells which release them
Provide a basic outline of the blood clotting cascade
Identify the events which trigger the commencement of blood clotting through the intrinsic and extrinsic pathways
- 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
Outline the purpose of the blood clotting cascade
- Both pathways activate Factor X (common endpoint)
- Thrombin is subsequently activated
- Thereafter, a fibrin clot is formed
Describe the modular structure of prothrombin
- 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
State the role of the Gla domain in blood clotting
The Gla domain allows interaction with sites of damage and brings together clotting factors
Describe the calcium-binding region of prothrombin
- 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
How is a fibrin clot formed?
⇒ 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
How is the fibrin clot stabilised?
- 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
Identify and describe three means of regulating the blood clotting process
- 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
What is classic haemophilia?
- 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
State the 7 key control points in blood clotting (formation & regulation)
- 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
