11. Regulation Of Protein Function Flashcards
What are isoenzymes?
Different forms of the same enzyme. They catalyse the same reaction but have different kinetic properties such as Km.
Hexokinase and glucokinase are examples of this.
What is product inhibition?
When the accumulation of the product of a reaction inhibits the forward reaction.
E.g Glucose-6-phosphate inhibits glucokinase activity.
What type of relationship between rate and substrate concentration do allosteric enzymes show?
Sigmoid relationship.
This is different to the rectangular hyperbola seen for simple enzymes.
What is meant by an allosteric enzyme?
Allosteric enzymes are enzymes that change their conformational ensemble upon binding of an effector, which results in an apparent change in binding affinity at a different ligand binding site.
What are the 2 different conformations that multi-subunit enzymes exist in?
T state- low affinity
R state - high affinity
What is the difference between allosteric activators and inhibitors?
They both bind to the enzyme at a site away from the active site and cause a conformational change
Allosteric activators increase the proportion of enzyme in the R state
Inhibitors increase the proportion of the enzyme in the T state
What are the key 2 enzymes involved phosphorylation and dephosphorylation?
Protein kinases- transfer the terminal phosphate from ATP to the -OH groups of Ser, Thr,Try.
Protein phosphotases - reverse the effects by catalysing the hydrolytic removal of phosphoryl groups from proteins.
Why is protein phosphorylation effective in altering enzyme activity?
It can completely change the enzyme conformation as it adds 2 negative charges and a phosphoryl group that can make H-bonds.
Glycogen breakdown and synthesis are reciprocally regulated. What does this mean?
It means that the signals that initiate the breakdown of glycogen will also inhibit glycogen synthesis.
How can enzymes be regulated in the long term?
- Change in the rate of protein synthesis - enzyme induction/repression
- Change in the rate of protein degradation - ubiquitin-proteasome pathway
What are allosteric activators and inhibitors for the phosphofructokinase enzyme in glycolysis?
Activators - AMP and fructose-2,6-bisphosphate
Inhibitors- ATP, Citrate, H+
What 3 amino acids do tyrosine kinases phosphorylate?
Serine, Threonine and Tyrosine
What enzyme reverses phosphorylation?
Protein phosphotases
How does the addition of a phosphate group alter the properties of an enzyme?
It adds 2 negative charges to the protein, which can cause conformational change.
Phosphoryl group can make additional hydrogen bonds
How are inactive zymogens activated?
Proteolytic cleavage
Give an example of enzymes which are synthesised as zymogens.
Digestive enzymes
Which digestive enzyme is the ‘master regulator’ of further activation of enzymes?
Trypsin
How does trypsin act as the master regulator of digestive enzymes?
It activates other zymogens, for example chymotrypsinogen to chymotyrpsin. Proelastase becomes elastase and prolipase becomes lipase.
How is trypsin activity controlled?
Pancreatic trypsin inhibitor binds to the enzyme tightly and stops its activity.
What is alpha1-antitrypsin?
A plasma protein that inhibits a range of proteases
How does a deficiency in alpha1-antitrypsin affect the lungs?
It is responsible for inhibiting elastase in the lungs, in a1at deficiency, elastase is not inhibited and it breaks down elastin, destroying alveoli lungs and can cause emphysema.
How can enzymes be regulated in the long-term?
- Change in rate of protein synthesis
2. Change in the rate of protein degradation
What are the 2 pathways which can stimulate the blood clotting cascade?
intrinsic pathway and extrinsic pathway
What does activation of either pathway lead to?
Factor X activation, common endpoint
Following factor X activation, how does a clot begin to form?
Factor X activates thrombin (from pro-thrombin) and converts fibrinogen to fibrin which then cross-links to form a fibrin clot
What enables many of the clotting factor proteins to activate others?
They are endopeptidases, so can chop peptide bonds in other clotting factors to activate zymogens into active enzymes.
What is the benefit of the clotting cascade?
It allows formation of a clot from activation of very small amounts of initial factor - signal amplification.
Outline the extrinsic activation pathway in the blood clotting cascade.
Upon vascular damage, tissue factor III is exposed, which cleaves factor VII to VIIa which then activates X to Xa.
Outline the intrinsic pathway of the blood clotting cascade.
XII to XIIa, XI to XIa, IX to IXa which then activates X to Xa.
12,11,9,10
How are factor IX and X targeted to the damaged membrane?
Gla domains
What is required for sustained thrombin activation?
Calcium ions which have a ‘bridging effect’
What is the molecular structure of prothrombin?
Gla domain, 2 kringle domains, and serine protease (thrombin part) at the C terminal.
What is the function of the 2 kringle domains in prothrombin?
They keep prothrombin in the inactive form
What is the function of the Gla domain at the N terminus of prothrombin?
It targets it to the appropriate site of action
When are Gla residues added onto the tissue clotting factors?
Added during post-translational modification of factors II,VII,IX and X in the liver.
What is the chemical basis of a Gla residue?
Addition of COOH groups to glutamate residues to form carboxyglutamate (Gla).
What charge do Gla residues have?
Negative (COOH)
If the cell membrane is damage, it will expose negative charges. How do the negatively charged Gla domains bind?
Calcium ions act as a cross-bridge, bind to membrane and clotting factors to allow interaction with sites of damage and bring together clotting factors.
What is the importance of Gla residues on prothrombin?
Prothrombin will bind calcium ions at the damaged membrane, which means that only prothrombin at the site of damage will be activated… clot will be localised to the site of damage.
Outline the structure of fibrinogen.
2 sets of tripeptides - alpha, beta and gamma
Joined at the N termini by disulphide bonds
3 globular domains linked by rods
What is the function of the N-terminal of fibrinogen?
N terminal of alpha and beta chains are highly negatively charged and prevent aggregation of fibrinogen.
Where is Trypsin secreted?
Pancreas
What protein inhibits trypsin function?
Pancreatic trypsin inhibitor
Describe how fibrin forms a soft/fibrin mesh clot.
Thrombin cleaves the alpha and beta chain at the central globular domain. Beta and Gamma chains at the C terminal ends interact with newly exposed groups at the cleaved N terminal, forming a soft clot, or a mesh clot.
Polymerisation at the C terminus.
Explain how a soft clot becomes a hard fibrin clot?
The soft clot is cross-linked with amide bonds on the side chains of lysine and glutamine residues.
Which enzyme is involved in hard clot formation and how is it activated?
transglutaminase, it is activated from protransglutaminase by thrombin.
What tissue factor is deficient in Haemophilia A?
VIII, 8
What is the result of the deficiency in haemophilia A?
VIII is not a protease itself, but it is an allosteric regulator of factor IXa which then activates factor X to form a clot. Hence the clotting process will be slowed.
How are proteases digested after a clot has formed?
Factors Va and VIIIa are degraded by protein C, which is activated when thrombin binds to the endothelial receptor thrombomodulin.
What can defects in Protein C cause?
Thrombotic disease
Name a specific thrombin inhibitor and how its activity may be increased.
Antithrombin 3 binds tightly to thrombin and inhibits its activity, it is enhanced by heparin binding.
Outline the process of fibrinolysis.
Plasminogen is activated by streptokinase or by tissue plasminogen activator, to plasmin. Plasmin then breaks down fibrin into fragments to dissolve the clot.
What is the difference between hexokinase and glucokinase activity?
Hexokinase has a high affinity for glucose and is constantly active.
Glucokinase has a low affinity and is only active when glucose levels peak after feeding.
