L15a- Protein regulation Flashcards
List the a) Short term and b) long term regulatory mechanisms that control enzyme activity
a) Short term:
1. Substrate and product concentration
2. Change in enzyme conformation: allosteric regulation, covalent modification + proteolytic cleavage
b) Long term:
1. Change in rate of protein synthesis
2. Change in rate of protein degradation
Explain how a) substrate concentration and b) product concentration can regulate enzyme activity
a) - Substrate concentration directly affects the rate of enzyme activity
- isoenzymes are different forms of the same enzyme that have different kinetic properties (can catalyse same reactions)
- some coenzymes have limited availability e.g. NAD/NADH
b) Accumulation of the product of a reaction inhibits forward reaction
Give an example of product inhibition in glycolysis
Glucose-6-Phosphate inhibits hexokinase activity
What are some characteristics of Allosteric enzymes
- they show a sigmoidal relationship between rate and [substrate]
- they are multisubunit enzymes
- can exist in 2 different conformations: T state (low affinity) + R state (high affinity)
- substrate binding to one subunit makes subsequent binding to other units progressively easier
a) Define allosteric regulation
b) What do allosteric i) activators and ii) inhibitors do? (Use the T and R state model)
a) Binding of a molecule away from the active site, causes an effect on enzyme activity
b) i) Activators: increase the proportion of enzyme in the R state (high affinity state), increasing the ROR
ii) increase the proportion of enzyme in the T state (low affinity state), decreasing the ROR
What would a graph of the following look like:
a) Allosteric enzyme without activator or inhibitor
b) Same enzyme w/ allosteric activator
b) Same enzyme w allosteric inhibitor
a) Sigmoidal curve
b) Shift curve to the left (Increase ROR)
c) Shift curve to the right (decreases ROR)
Phosphofructokinase-1 is a key enzyme in glycolysis. It is allosterically regulated.
a) What does it do?
b) List the allosteric i) activators and ii) inhibitors of this enzyme related to the T/R model and
a) Catalyses the conversion of Fructose-6-Phosphate into Fructose 1,6-bisphosphate using ATP
b) i) Activators:
- [AMP]: It is a low energy signal, activating PFK-1 to increase its activity, increases proportion of enzyme in R (high affinity) state
- Fructose-2,6-Bisphosphate: made by PFK-2 from F-6-P, will bind to PFK-1 to stabilise the high affinity (R) state to increase activity
ii) Inhibitors:
- ATP: high energy signal, will bind to regulatory sites lead to conformational change, stabilising low affinity (T) state of enzyme to decrease activity
- Citrate: stabilises the T state of the enzyme
- H+: Stabilises T state of the enzyme to control acidity
Which enzymes:
a) Phosphorylate
b) Dephosphorylate
How?
a) Protein kinases- transfer the Po43- from ATP to the -OH group Ser, Thr + Tyr
b) Protein phosphatases- reverse the effects of kinases by catalysing the hydrolytic removal of phosphoryl groups from proteins
Why is protein phosphorylation so effective?
- Adds 2 -ve 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
a) What is amplification by enzyme cascades ?
b) Why is it important ?
a) - Enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade
b) allows amplification of the initial signal by several orders of magnitude within a few milliseconds
How are some enzymes activated ?
- specific proteolytic cleavage: breaking peptide bonds
Examples of proteolytic cleavage?
- Digestive enzymes synthesised as zymogens in stomach and pancreas e.g. pepsinogen, trypsinogen
- Some protein hormones e.g. insulin are synthesised as inactive precursors
- Blood clotting is mediated by cascade of proteolytic activations
- Programmed cell death is mediated by proteolytic enzymes
Examples of long term regulation of proteins?
- Change in rate of protein synthesis: enzyme induction/repression
- Change in rate of protein degradation; ubiquitin-proteasome pathway
What are the steps of the blood clotting cascade?
2 pathways that lead into it either:
a) Intrinsic pathway: Damaged endothelial lining of blood cells promotes binding of factor XII
b) Extrinsic pathway: trauma releases tissue factor (factor III)
Then:
- Factor X activation: common end point for both pathways
- Thrombin activation
- Formation of fibrin clot
Why is a cascade necessary for blood clotting?
- allows rapid formation of a clot from activation of very small amounts of the initial factor
