How are enzymes regulated long term?
- Change in rate of protein synthesis
2. Change in rate of protein degradation
How are enzymes regulated short term?
- Substrate and product concentration
2. Change in enzyme conformation
How can you change the conformation of an enzyme (linked to short term regulation)?
- Allosteric regulation
- Covalent modification
- Proteolytic cleavage
What is the blood clotting cascade?
- Intrinsic pathway (damaged endothelial lining) and extrinsic pathway (trauma)
- Factor X activated
- Thrombin is activated (prothrombin —> thrombin)
- Formation of a fibrin clot (fibrinogen —> fibrin)
What does the blood clotting cascade allow for?
Allows the formation of a clot from activation of very small amounts of the initial factor
What are the domains in prothrombin? What is their role?
- 2 Kringle domains - help keep prothrombin in the inactive form
- Gla domain - target the protease to the appropriate site for activation
How does a fibrin clot form?
- Thrombin cleaves the fibrinopeptides A and B from the central globular domain of fibrinogen
- The globular domains at the C-terminal of the beta and gamma chains interact with the exposed sequences at the N-termini of the cleaved B and A chains
- Forms a fibrin mesh/clot
- Stabilised by the formation of aside bonds between lysine and glutamine residues (catalysed by (protransglutaminase —>) transgulatminase)
Which factor has a defect that causes haemophilia?
Factor VIII (antihaemophilic factor)
What type of feedback does thrombin cause?
*an increase in thrombin leads to an increase in factor XIa, VIIIa, Va and XIIIa
How is the clotting process stopped?
- Localisation of (pro)thrombin
- Digestion by proteases
- Specific inhibitors
How does localisation of (pro)thrombin stop the clotting process?
Leads to dilution of clotting factors by blood flow and are removed by the liver
How does digestion by proteases stop the clotting process?
Degradation by proteases activated by thrombin binding to thrombomodulin receptor (negative feedback)
E.g. protein C degrades factors Va and VIIIa
(Defects in protein C can cause thrombotic disease)
How do specific inhibitors stop the clotting process?
An example of an inhibitor is antithrombin III (AT3) which is enhanced by heparin binding
How is the blood clot removed?
Plasminogen —> plasmin (activated by streptokinase and t-PA)
Plasmin breaks down fibrin to fibrin fragments
What are the 7 key control points in blood clotting?
- Inactive zymogens present at low concentration
- Proteolytic activation
- Amplification of initial signal by cascade mechanism
- Clustering of clotting factors at site of damage
- Feedback activation by thrombin ensures continuation of clotting
- Termination of clotting by multiple mechanisms
- Clot breakdown controlled by proteolytic activation
How does the substrate and product concentration regulate enzyme activity?
- substrate availability will affect the rate of enzyme activity
- isoenzymes are different forms of the same enzyme (so have different kinetic properties/parameters e.g. hexokinase and glucokinase)
- accumulation of product inhibits the forward reaction (e.g. G-6-P inhibits hexokinase activity)
How does allosteric regulation regulate enzyme activity?
Shows a sigmoidal relationship with T and R states (T - low affinity, R - high affinity)
Substrate binding to one subunit makes subsequent binding to other subunits easier
What are allosteric activators and allosteric inhibitors?
Activators - increase the proportion of enzyme in the R state
Inhibitors - increase the proportion of enzyme in the T state
What is an example of allosteric regulation?
Phosphofructokinase (PFK) is allosterically regulated
Activated by AMP, fructose-2,6-bisphosphate (low energy)
Inhibited by ATP, citrate and H+ (high energy)
How does covalent modification regulate enzyme activity?
- protein kinases - add phosphate Fromm ATP to OH group of Ser, Thr, Tyr
- protein phosphatases - reverse the effects of kinases by hydrologic removal
Why is adding a phosphate effective?
- adds a negative charge
- can make H bonds
- allows for amplification
- rate of phosphorylation/dephosphorylation can be adjusted
Describe the process of proteolytic activation of chymotrypsinogen.
Trypsin activates chymotrypsin from chymotrypsinogen
What is zymogen activation by proteolytic cleavage?
Enteropeptidases activate trypsin (from trypsinogen)
How do you inhibit proteolytic cleavage?
With endogenous inhibitors
E.g. pancreatic trypsin inhibitor binds to trypsin and stops the activity
How are enzymes activated by proteolytic cleavage?
- Digestive enzymes are synthesised from zymogens
- Protein hormones are synthesised as inactive precursor e.g. insulin
- Blood clotting is mediated by a cascade of proteolytic activations
- Many developmental processes are controlled by activation of zymogens
- Apoptosis is mediated by proteolytic enzymes (capsases)