Session 8 ILOs - Protein function & regulation Flashcards
List the major short term mechanisms that regulate enzyme activity (plus examples)
- Isoenzymes (different enzyme forms) e.g. Hexokinase & Glucokinase in the liver
- Allosteric regulation (change in enzyme conformation) e.g. 2,3 BPG increases the stabilisation of the T state of haemoglobin
- Phosphorylation (reversible covalent modification) e.g. protein kinases & protein phosphotases
- Proteolytic activation e.g. trypsinogen to trypsin
Explain T & R states of enzymes
R state (relaxed state) increases affinity for substrate T state (tense state) decreases affinity for substrate
What are the allosteric properties of a key regulatory enzyme?
Allosteric enzymes have an additional site for an effector to bind to, in addition to the active site
Usually multi-subunit enzymes
Can exist in 2 different forms (R or T state) so either act as:
1. Allosteric activators = increase proportion of enzymes in the R state
2. Allosteric inhibitors = increase proportion of enzymes in the T state
Why are cascades important?
Cascades allows certain processes to be tightly controlled (IRREVERSIBLE)
e.g. used in blood clotting, digestive enzymes, apoptosis
What is a kinase/phosphotase?
Protein kinases: transfer terminal phosphate from ATP to the OH- group of Serine/Threonine/Tyrosine
Protein phosphatases: reverses the effects of the kinases by catalysing the hydrolytic removal of the phosphoryl groups from proteins
Why does phosphorylation have an effect?
The free energy of phosphorylation is large, it adds 2 -ve charges and a phosphoryl group can make H bonds
Links energy status of the cell to metabolism through ATP
Allows for amplification effects
Define the term zymogen/proenzyme (plus examples)
A zymogen or proenzyme is an inactive precursor molecule
They can be activated by breaking of a peptide bond which removes the ‘pro’ segment in an irreversible reaction
EXAMPLE: In terms of digestive enzymes, Trypsinogen is converted to Trypsin (by Enteropeptidase) in the gut which then goes on to cleave other digestive enzymes in the zymogen state
Explain how activation of the clotting cascade leads to the formation of fibrin
Damage (intrinsic or extrinsic) leads to a series of reactions, each catalysed by an enzyme, which causes factor X activation of Prothrombin to Thrombin which then catalyses the conversion of Fibrinogen to Fibrin (cross-linked fibrin clot)
Thrombin cuts off the fibriopeptides off the fibrinogen which leads to soft clots. Further cross linking by covalent bonds catalysed by transglutaminase, leads to a more solid clot
Outline the mechanisms involved in regulation of clot formation
Post-translational modification of factors 2, 7, 9, 10 in the liver.
At the site of injury, negatively charged phospholipid regions are exposed, which attract calcium ions which are positively charged. Gamma-carboxyglutamate residues can be made from glutamate (in the liver), which add negative charges and are attracted to the calcium ions. This allows interaction with sites of damage and it brings together clotting factors.
Outline the mechanisms involved in regulation of clot breakdown
- Localisation of (pro)thrombin - dilute the clotting factors by blood flow and help by the liver
- Digestion by proteases - some factors are degraded by protein C
- Binding of specific inhibitors e.g. anti-thrombin 3 (stop clotting factors working and they get degraded in the liver)
Fibrinolysis - plasminogen is converted to plasmin, activated by t-PA or streptokinase. Plasmin converts fibrin to fibrin fragments which breaks down the clot structure
Explain the roles of haemoglobin & myoglobin
Haemoglobin: haemoglobin is an oxygen transport protein found in RBCs
Myoglobin: monomeric protein found mainly in muscle tissue serving as an intracellular storage site for oxygen
Contrast the oxygen binding properties of haemoglobin & myoglobin plus why haemoglobin is most suited to its role as an oxygen transporter
Haemoglobin has 4 subunits with 4 haem groups per molecule, whereas myoglobin has 1 unit with 1 haem group.
Haemoglobin has a sigmoidal curve and an ability to change its affinity for oxygen depending on the environment = cooperative effect due to the 4 subunits. Monomeric myoglobin can’t change it’s conformation in the same way
Describe the major structural differences between oxygenated and deoxygenated haemoglobin and the molecular basis of cooperativity
The interaction of the O2 molecule with one heme molecule pulls the Fe2+ ions closer to the ring which changes its shape. Ionic interactions holding the 4 chains together are distracted and as they reform in a different position, the quaternary structure is altered which increases the binding affinity
Describe the effect of CO2, H+ ions, 2,3-BPG and carbon monoxide on the binding of O2 to haemoglobin
Increased CO2: decrease in the binding affinity
Increased H+: decrease in the binding affinity
Increased 2,3-BPG: decrease in the binding affinity
Increased Carbon monoxide: decrease in the binding affinity (competitive inhibitor)
Can lead to more offloading of oxygen (good in respiring tissues, not good in the lungs!)
Describe the mutation in sickle cell anaemia and why this causes sickle cell anaemia
Mutation of Glutamate to Valine in Beta-globing unit - causes the subunit to reorientate itself and valine interacts with hydrophobic patches on other haemoglobin molecules = molecules stick together
Also more rigid & more likely to lyse
Outline key features needed for protein secretion
Proteins for cytosol / imported into organelles are synthesised on free ribosomes
Proteins for the membrane or secretory pathway are synthesised by ribosomes in the rough ER
Name the differences between constitutive and regulated secretion
Constitutive secretion goes on all the time and doesn’t need to be altered day to day EXAMPLE: Albumin
Regulated secretion only occurs when needed by 3 cells:
Endocrine cells: secreting hormones
Exocrine cells: secreting digestive juices
Neurocrine cells: secreting neurotransmitters
Outline the structure of collagen
The triple-helical structure of collagens consists of three distinct α-chains, with a glycine in every 3rd position in each chain and mostly proline/hydroxyproline in the other positions. This allows h-bonds to form between alpha chains to stabilise structure
Why is collagen structurally very stable?
Lysyl oxidase forms covalent bonds between lysine residues and hydrogen bonds between chains stabilises the structure
Outline the processing of collagen
- Chain is synthesised and enters the lumen of the RER
- Cleavage of signal peptide by the signal peptidase
- Hydroxylation of selected proline and lysine residues (adding OH)
- Addition of N-linked oligosaccharides
- Addition of galactose to hydroxylysine residues
- 3 chains align and disulphide bonds form in C-terminus regions
- Formation of triple-helical pro collagen from C- to N-terminus
- More glycosylation reactions (adding glucose) complete the O-linked oligosaccharide chains
- Transported into transport vesicle ready for release
SEE NOTES
Explain the role of Prolyl Hydroxylase in collagen formation
Prolyl Hydroxylase allows increased H-bonding to stabilise the triple helix by addition of hydroxyl groups
Requires vitamin C & Fe2+ so if individual has a vitamin C deficiency, then weak tropocollagen triple helices result and they develop scurvy
