Regulation of Protein Function Flashcards
How are enzymes regulated in the short term?
Substrate/product concentration and change in enzyme conformation (allosteric regulation, covalent modification, proteolytic cleavage)
How are enzymes regulated in the long term?
Change in rates of protein synthesis and degradation
What will substrate availability have an effect on?
Rate of enzyme activity
What are isoenzymes?
Different forms of the same enzyme that have different kinetic properies (catalyse same reaction)
What is product inhibition?
Accumulation of the product of a reaction inhibits the forward reaction
What relationship do allosteric enzymes show between rate and substrate concentration?
Sigmoid
What relationship do simple enzymes show between rate and substrate concentration?
Rectangular hyperbola
What are allosteric enzymes?
Enzymes that change their conformation upon binding of an effector
What are the two states that multi-subunit enzymes can exist in?
T state - low affinity
R state - high affinity
What is the effect on subsequent binding of a substrite binding to one subunit of an enzyme?
Substrate binding to one subunit makes subsequent binding to other subunits progressively easier
What do allosteric activators do?
Increase the proportion of enzyme in the R state (increase affinity)
What do allosteric inhibitors do?
Increase the proportion of enzyme in the T state (decrease affinity)
What does a shift to the left of the graph show in terms of affinity?
Shift to left = higher affinity = increased proportion of enzyme in R state
Do activators shift the graph to the left or right?
Left
What enzyme sets the pace for glycolysis?
Phosphofructokinase - it is allosterically regulated
What are some allosteric activators of phosphofructokinase?
AMP, fructose-2,6-bisphosphate
What are some allosteric inhibitors of phosphofructokinase?
ATP, citrate, H+
What can protein kinases do to serine, threonine and tyrosine?
Transfer the terminal phosphate from ATP to the OH group of these amino acids
What do protein phosphatases do?
Reverse the effects of kinases by catalysing the hydrolytic removal of phophoryl groups from proteins
Why is protein phosphorylation so effective?
Adds 2 negative charges, 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
What can the two negative charges added to proteins via phosphorylation do?
Cause conformational change
What is the difference between a phosphoryl group and a phosphate?
Phosphate = PO4(^3-) Phosphoryl = PO3 (^2-)
What is a zymogen?
An inactive substance which is converted into an enzyme when activated by another enzyme (precursor to enzymes)
How is apoptosis mediated?
Proteolytic enzymes (caspases) which are synthesised in inactive (procaspase) form - only activated when there is a covalent breakage of peptide bond
What is the ‘master regulator’ of the activation of enteropeptidases?
Trypsin
a1-antitrypsin inhibits a range of proteases. What is a disease that involves the deficiency of a1-antitrypsin?
Emphysema - destruction of alveolar walls by elastase
What do both the intrinstic and extrinsic pathways of the blood clotting cascade lead to?
Factor X activation, which leads to thrombin activation and the formation of fibrin clot
What is factor X?
Endopeptidase that contains Gla residues. Also known as the Stuart-Prower factor.
What is the activated form of factor X?
Factor Xa, activated by hydrolysis
How does factor X act?
Cleaves prothrombin in two places which yields the active thrombin
What does thrombin do?
Converts fibrinogen into fibrin
What plays a role in the activation of the intrinsic pathway?
Membrane damage
In the intrinsic pathway, what are targeted to membrane by Gla domains?
Factor IX and X
What helps keep prothrombin in the inactive form?
The two kringle domains
In which domain of prothrombin is the protease function (thrombin part) contained?
C-terminal
What targets prothombin to appropriate sites for its activation?
Gla domains
What is Gla?
g-carboxyglutamate
gamma
What is the role of Gla residues?
Post translational modification of factors I, VII, IX, X in the liver, addition of COOH groups to glutamate residues which form Gla and allows interaction with sites of damage and brings together clotting factors
What does prothrombin bind to?
Calcium ions via Gla residues
How are clots localised to the site of damage?
Only prothrombin next to the site of damage will be activated
What is the structure of fibrinogen?
340 kDa protein
2 sets of tripeptides joined at N termini by disulphide bonds
3 globular domains linked by rods
What prevents aggregation of fibrinogen?
N terminal regions of alpha and beta chains are highly negatively charged
How is a fibrin clot formed?
Thrombin cleaves fibrinopeptides from the central globular domain of fibrinogen. Globular domains at the C terminal ends of the b and g chains interact with exposed sequences at the N termini of the cleaved b and a chains to form a fibrin clot
What is the newly formed clot stabilised by?
Formation of amide bonds between side chains of lysine and glutamate residues
What is the cross linking reaction in formation of blood clots catalysed by?
Transglutaminase
What is classic haemophilia a defect in?
Factor VIII
What is factor VIII and what is it increased by?
It is not a protease but stimulates the activity of factor IX which is. It is increased by limited proteolysis by thrombin and factor Xa. This positive feedback amplifies the clotting signal and accelerates clot formation
How is the clotting process stopped?
Localisation of prothrombin - dilution of clotting factos by blood flow and removal by liver.
Digestion by proteases
What is protein C?
Degrades factors Va and VIIIa. Activated by thrombin binding to thrombmodulin.
What can defects in protein C cause?
Thrombotic disease
How can the clotting process be regulated?
Specific inhibitors - antithrombin III
What are some key control points in blood clotting?
Inactive zymogens present at low concentration, proteolytic activation, clusetering of clotting factors and site of damage, feedback activation by thrombin ensures clotting continuation, clot breakdown by proteolytic activation
How can clot formation be reversed?
Proteolytic cleavage of fibrin by the enzyme plasmin
