Week 3- Regulation of Protein Activity Flashcards
Long-term regulation of protein activity
Change in rate of protein synthesis: enzyme induction/repression
Change in rate of protein degradation: proteasome-ubiquitin pathway
Isoenzymes
Enzymes that catalyse the same reaction but have a different amino acid sequence. They may also have different kinetic properties.
Product inhibition of enzymes
Accumulation of the product of a reaction inhibits forward reaction.
For example: glucose-6-phosphate inhibits hexokinase activity
Allosteric regulation of enzymes
- Sigmoid relationship between rate and substrate concentration
- Multi-subunit enzymes
- Can exist in two conformations
- Substrate binding to one subunit makes subsequent binding to other subunits progressively easier
Allosteric activators and inhibitors
Activators: increase proportion of enzyme in R state
Inhibitors: Increase proportion of enzyme in T state
Allosteric regulation of phosphofructokinase
Fructose-6-phosphate + ATP –> Fructose-1,6-bisphosphate + ADP + H+
-Phosphofructokinase sets the pace for glycolysis.
Activators of phosphofructokinase: AMP, Fructose-2,6-bisphosphate
Inhibitors of phosphofructokinase: ATP (high-energy signal), Citrate, H+
Zymogen
The inactive precursor of a proteolytic enzyme
Covalent modification of enzymes
- Phosphorylation
Protein kinases
Add phosphate groups to particular amino acid residues that have hydroxyl groups;
Transfer terminal phosphate of ATP to hydroxyl group of Serine, Threonine and Tyrosine;
Protein phosphatases
Reverse effects of kinases by catalysing hydrolytic removal of phosphoryl groups from proteins
List the digestive enzymes synthesised as zymogens in the stomach and pancreas.
Stomach- Pepsinogen (Pepsin)
Pancreas - Chymotrypsinogen (chymotrypsin), Trypsinogen (trypsin), Procarboxypeptidase (carboxypeptidase), Proelastase (elastase)
Caspases
Proteolytic enzymes which are synthesised in inactive form and mediate programmed cell death (apoptosis)
Give examples of endogenous inhibitors that regulate protease activity.
- Pancreatic trypsin inhibitor: binds trypsin and stops activity
- alpha1-antitrypsin: 53 kDa plasma protein that inhibits a range of proteases
Emphysema
- Deficiency of alpha1-antitrypsin
- Destruction of alveolar walls by elastase
Short-term regulation of enzyme activity
- Substrate and product concentration
2. Change in enzyme conformation: allosteric regulation, covalent modification, proteolytic cleavage
Extrinsic pathway of blood clotting
- Activation of factor VII: membrane damage exposes extracellular domain of tissue
- Autocatalytic activation of factor III
Intrinsic pathway of blood clotting
- Membrane damage plays a role in activation of intrinsic pathway
- Factor IX and X are targeted to membrane by gla domains
- Ca2+
- Required for sustained thrombosis activation
Activation of thrombin
- Proteolytic cleavage at Arg 274 to release a fragment containing first three domains
- Cleavage after Arg 323 releases fully active thrombin consisting of two chains, 6 kDa and 31 kDa linked by disulphide bonds.
Role of carboxyglutamate (Gla) residues:
- Post-translational modification of factors II, VII, IX, X in the liver
- Addition of COOH groups to glutamate residues to form carboxyglutamate
- Allows interaction with sites of damage and brings together clotting factors
- Calcium can act as a cross bridge and attract negatively charged carboxyl groups on clotting factors
- Process: vitamin K dependent
Calcium binding region of prothrombin
- Prothrombin binds calcium via Gla residues
- Only prothrombin next to site of damage will be activated
- clots localised to site of damage
Structure of fibrinogen
- 340 kDa protein
- 2 sets of tripeptides (alpha, beta and gamma) joined at N-termini by disulphide bonds
- 3 globular domains linked by rods
- N-terminal regions of alpha and beta chains are highly negatively charged: prevent aggregations of fibrinogen
Formation of fibrin clot
- Thrombin cleaves fibrinopeptides A and B from central globular domain of fibrinogen
- Globular domains at C-terminal ends of B and Y chains interact with exposed sequences at N-termini of cleaved beta and alpha chains to form fibrin mesh
- The newly formed clot is stabilised by the formation of amide bonds between 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.
Classic hemophilia
- Defect in factor VIII
- Factor VIII stimulates activity of factor IXa (a serine protease)
- Activity of factor VIII markedly increased by limited proteolysis by thrombin and factor Xa. This positive feedback amplifies clotting signal and accelerates clot formation.
- Treatment with recombinant factor VIII
Ways to stop the clotting process
- Dilution of clotting factors by blood flow and removal by liver
- Digestion by proteases: for example, factor Va and factor VIIIa are degraded by protein C; protein C is activated by thrombin binding to endothelial receptor, thrombomodulin
Defects in protein C –> thrombotic disease - Specific inhibitors: antithrombin III binds strongly to factor X: enhanced by heparin binding, AT3 does not act on thrombomodulin bound thrombin
