Lecture 8 Flashcards
Lists regulatory mecahisms in the cell
- Extracellular signal
- Transcription of specific genes
- mRNA degradation
- mRNA translation on ribosome
- Protein degradation
* *6. Enzyme sequestered in subcellular reticulum - Enzyme binds substrate
- Enzyme binds ligand (allosteric regulation)
- Enzyme undergoes phosphorylation/dephosphorylation
- Enzyme combines with regulatory protein**
List 4 examples of Short term regulation
‘GAPPI’
- Different enzyme forms - isoenzymes
- Change in enzyme conformation – allosteric regulation
- Reversible covalent modification – phosphorylation
- Proteolytic activation
- Controlling the amount of enzyme present – gene expression
Different enzyme forms - isoenzymes (definition of this, what does this mean for regulation?, ways to get isoenzymes, )
Definition: Enzymes that catalyse the same reaction but have different amino acid sequence (similar active site. as it can catalyse the same reaction)
- Different activity
- Different regulatory properties
- Synthesised from the different genes
- differentially spliced from same gene
Example of isoenzymes
2 isoenzymes catalysing the phosphorylation of glucose:
- hexokinase has a very low km value i.e. very high affinity
- glucokinase has a much higher km value i.e. lower affinity
Reason - In starvation, higher glucokinase activity compared to fed state, as trying to maintain some glucose circulation
2nd regulatory mechanism
- Change in enzyme confomration - allosteric regulation (what is allosteric regulation, what does this mean?, graph to show the states)
Action at a distance i.e. binding elsewhere on the enzyme at an allosteric site (away from the active site). Results in conformational changes.
Allosterically regulated enzymes (i.e. one enzyme that exists in two states/two different forms:
• Usually multi-subunit
• Can exist in 2 different forms
- T state = low affinity (T state - less affinity for the substrate)
- R state = high affinity
Do not obey Michaelis-Menten kinetics
Curve in the middle - there will be a mixture of enzymes in each state at any given time, so will see a combination of the two curves (curve in the middle)
What are allosteric effectors?
Allosteric effector: is a molecule that binds to an enzyme and causes allosteric effects. Positive allosteric effects involve “activation” of the enzyme - increasing its activity. Negative allosteric effects involve “inhibition” of the enzyme - decreasing its activity.
Allosteric regulation (how to increase proportion enzymes in each state)
Activators - Increase the proportion of enzyme in the R state (high affinity form increases activity)
Inhibitors - Increase the proportion of enzyme in the T state (low affinity form decreases activity)
Specific example of allosteric regulation
Example of an allosterically regulation enzyme -
Phosphofructokinase-1:
Activators: (allosteric activators)
- AMP
- fructose 2,6-bisphosphate
Inhibitors: (allosteric inhibitors)
- citrate
- ATP
- H+
3rd regulatory modification: Reversible covalent modification - phosphorylation (what is this, example,
Modified by the addition of different groups, like adding a phosphate group (this is form of allosteric modification)
Example: Protein with serine side chain -
Protein kinases
transfer the terminal phosphate from ATP to the –OH group of Ser, Thr, Tyr
Protein phosphatases
reverse the effects of kinases by catalysing the hydrolytic removal of phosphoryl groups from proteins. (reversibility)
Reversible covalent modification - phosphorylation - (why so effective, what this does, the effect of this, rate, energy in cell, amplifcation)
- Why is protein phosphorylation so effective?
- The free energy of phosphorylation is large (involves hydrolysis of ATP - lots of free energy released)
- Adds 2 negative charges (disrupt or enhance interactions between and within subunits, or with other regulatory molecules)
- A phosphoryl group can make H-bonds (allows different interactions between and within subunits, or with other regulatory molecules)
- Rate of phosphorylation/dephosphorylation can be adjusted
- Links energy status of the cell to metabolism through ATP (think about ‘high energy status’, ‘low energy status’, ATP is a high energy status molecule)
- Allow for amplification effects (next slide)
Reversible covalent modification - phosphorylation AMPLICATION
When enzymes active enzymes, the number of affected molecules increases geometrically in an enzyme cascade.
This shows that a small signal, can lead to a large effect, very quickly
Pic - 9 fold amplification of our activity (in reality - thousands/millions amplification)
Proteolytic activation -
- Inactive precursor molecules (e.g. zymogens or proenzymes) that are completely inactive. They are inactive when they are in the zymogen or proenzyme form.
- The inactive precursor molecule has a different amino acid sequence to the final activated form
- How to go from an inactive form to an active form:
- take off part of the original proenzyme. Do this through breaking specific peptide bonds (removing the ‘pro-segment’)
- by breaking this peptide bond = now it is activated
- This is very important when processes need to be tightly controlled
- Irreversible
- Enzymes: blood clotting, digestive enzymes, apotosis (don’t want this to occur randomly, needs to be either ‘on’ or ‘off’ - nothing in between)
Zymogen definition
An inactive substance which is converted into an enzyme when activated by another enzyme.
Pancreatic zymogens (example of proteolytic activation)
Look at pic
Need to learn an example of zymogens
- E.g. trpsinogen is the inactive form of typsin (typsin is the active form). It will be turned on when it is secreted by the pancreatic duct. Enteropeptidase ‘cleaves’ (cuts) the specific peptide bond, this then turns it into it’s active trypsin molecule.
Trypsin - master regulator
When trypsin is activated, it will stimulate the activation of other zymogen and proenzymes present
What do I need to know?
• list the major regulatory mechanisms that control enzyme activity (plus examples).
• discuss the allosteric properties of a key regulatory enzyme.
- what is allostery?
- R and T states
- allosteric effectors
- give examples
• discuss the concept of enzyme cascades and the use of protein kinases and phosphatases to regulate activity.
- why cascades are important
- what is a kinase/phosphatase?
- why does phosphorylation have an effect?
• define the term zymogen (with examples).
Blood clotting cascade - must be tightly regulated! Summary of this
Blood clotting cascade -
Intrinsic and extrinsic pathway leads to thrombin activation. One active enzyme molecule, leads to activation of the next molecule….
A series of reactions each catalysed by an enzyme
Each step leads to an amplification of the original signal
Rapid amplification: Very small amounts of the initial signal needed to trigger formation of a clot, i.e. activation of one molecule, leads to the activation of 100 molecules, and these then all activate another 100 each… (helps clot to form quickly and in the right place)
Understanding the structure of prothrombin (then allows understanding of thrombin)
Pic - N terminal to left, C terminal to right
Proteolytic activation:
• The protease function (the thrombin part) is contained in the C-terminal domain. (this is the serine protease part)
• The two kringle domains help keep prothrombin in the inactive form
• Gla domains target it to appropriate sites for its activation (will go in more depth later in flashcards - but the gla is modified into y-carboxyglutamate residues, these binds to the Ca2+ on the endothelial surface, the prothrombin is then in close proximity to the clotting factors that stimulate proteolytic activation of prothrombin to thrombin)
Fibronogen is converted by thrombin into fibrin
Fibrinogen = precursor molecule
• Composed of 3 polypeptide chains – (A alpha-2 , B beta-2 , gamma-2 )
• 2 globular heads separated by rod-like triple-helical alpha helices
• Fibrinopeptides – prevent fibrinogen molecules coming together from the two seperate chains (otherwise this would start the formation of a clot, therefore, the fibronopeptides are stopping clots forming)
Pic - see the two strands in diagram above, central globular unit and the fibrinopeptides (see notes if confused - labelled here)
F stands for fibrinopeptides
2 means 2 strands
Fibrinogen converted by thrombin into fibrin
- Thrombin cuts of fibrinopeptides to produce fibrin (fibrinogen now able to come together)
- Fibrin monomers assemble by non-covalent interactions – “soft clot”
- Cross-linking of soft clot by covalent bonds between Lys and Gln residues – catalysed by transglutaminase (Factor XIII)
How is sustained activation of the pathway (blood clot pathway) sustained?
- Factors V and VIII – cofactors that stimulate activity of other enztmes in the pathway (cofactors enhance the activity in this part of the pathway, remember cofactors are called clotting factors)
- Thrombin – positive feedback on factors V, VIII and XI and XIII. Pathway is sustained once it has become activated. Don’t need the damage to the blood vessel to still be there -> positive feedback affect of thrombin.
NB: Thrombin has to be created by the original clot first, i.e. factors VII, Va, VIIIa, XIa, can’t be activated by thrombin at the beginning as thrombin would at this stage still be in prothrmobin form
Role of y-carboxyglutamate residues (Gla) in prothrombin
- Damage to lining of the endothelium layer will express the negative charge as the phospholipid heads are negatively charged
- Ca2+ will bind to the negatively charged phospholipids head (phosphate)
- Gla residue is negatively charged, it brings the prothrombin to the side of damage
- the clotting factor by proteolytic activation will activate the prothrombin as it causes the prothrombin to change into thrombin.
(i think - wait on email from Dr L)
or this is the case:
- y-carboxyglutamate is negatively charged so will also bind to the Ca2+. The y-carboxylate will just float around in the blood until there is a gap in the endothelium lining (like diagram below - but showing for clotting factors rather than prothrombin!)
- Attachment to membrane increases the rate at which the proteolytic conversion of prothrombin to thrombin can occur. i.e. the prothrombin binding to the Ca2+ by it’s y-carboxyglutamate residue, activates prothrombin -> thrombin
- Thrombin then causes the conversion of fibrogen into fibrin
Role of y-carboxyglutamate residues (Gla) with clotting factors
- The clotting factors in the blood are quite dilute, need them to be in high quantities at the site of damage
- Damage to the lining of the endothelial layer exposes negatively charged phospholipids
- Ca2+ can bind to these negatively charged phospholipids
- Clotting factors like II, VII, IV, X, all contain these Gla residues
- In the liver, the clotting factors are post-translationally modified, this is where the addition of carboxyl groups to glutamate residues makes the y-carboxyglutamate which is very negative
- The y-carboxyglutamate residues are attracted to the positive Ca2+ at the site of damage (like ‘magnets’)
- Therefore, this helps bring clotting factors to the site of damage
What vitamin dependent is post-translocation modification of precursors of II,VII,IX,V to y-carboxyglutamate II, VII, IX, X?
Addition of COOH groups to glutamate residues to form carboxyglutamte (Gla)
This requires vitamin K
Problem with trying to stop the clotting process
These methods have to get rid of the clotting factors, however, once activated they can’t be ‘unactivated’. They have to be removed and degraded.
Mechanisms to stop the clotting process
-
Localisation of (pro)thrombin
Dilution of clotting factors by blood flow, and removal by liver (Blood flow will remove the clotting factors II, VII, IX, X, then removed by the liver) -
Digestion by proteases - digest the clotting factors (can’t just add the pro-segment back again!)
- for example, factors Va and VIIIa are degraded by protein C
- protein C is activated by thrombin negative feedback loop (see pic below) -
Binding of specific inhibitors e.g. antithrombin III
- This binds extremely tightly to some of the clotting factors, this will stop them from working. Then are degraded in the liver.
Name of the process of breaking the clot
Fibrinolysis
How breaking the clot occurs?
Look at pic below
t-PA and Streptokinase can cause proteolytic activation of plasminogen
Plasminogen (inactive precursor)
Plasmin (active form)
Summary pic of oxyheamoglobin and carbaminoheamoglobin
How does oxygen bind to heamoglobin and myoglobin?
Oxygen binding changes the position of the iron ion Haem group is bound to the polypeptide chain via a histidine residue
1 iron per heam group
Binding of oxygen to myoglobin - graph curve, affinity, describe the curve
- Oxygen binding to myoglobin shows a hyperbolic dependence on oxygen concentration (hyperbolic dependence - reaction rate is almost linear at the beginning, however, as the substrate conc. is increased to higher and higher levels, the reaction no longer increase in the same proportion to the increase in substrate conc, starts to have a smaller and smaller increase in rate until asymptote)
- Constant affinity for oxygen (Binding of the oxygen is not affected by ppO2 as myoglobin is only one polypeptide chain
Binding of oxygen to heamoglobin (curve, affinity…)
- Oxygen binding in haemoglobin shows a sigmoidal binding curve
- Affinity for oxygen increase with the partial pressure for oxygen (binding changes with ppO2)
What happens to heamoglobin after one oxygen has bound?
Heamoglobin undergoes conformational change on the binding of oxygen
Changes to the structure changes the affinity of oxygen to it.
T state -> R state.
R state has a higher affinity, it is formed after one oxygen has bound. Binding of one oxygen acts as an allosteric effect. The binding of one oxygen changes the structure from deoxyhemoglobin to oxyhemoglobin.
- *- T state in deoxyheamoglobin
- R state in oxyheamoglobin**
- *Therefore oxyheamoglobin affinity is higher to bind to another oxygen compared to deoxyheamoglonin binding with it’s 1st oxygen.** This is positive cooperativity.
What is cooperativity? What does this mean for ‘states’? Graph showing the states etc?
Binding of oxygen promotes transition from low affinity T state to high affinity R state
Before binding to oxygen, heamoglobin is T state
After binding to oxygen, oxyheamoglobin then picks up another oxygen, has a higher affinity so it R state
Graph:
Get a mix between T-state binding curve and R-state binding curve.
Explanation of the T-state binding curve - e.g. at ppO2 150, the curve shows the affinity of deoxyheamoglobin -> oxyheamoglobin (with one oxygen)
The affect of ppO2 in lungs and in tissues on the saturation of hemoglobin
The sigmoidal properties of Hb allows greater oxygen transport compared to comparable proteins entirely in the high affinity R state or low affinity (T)
- Heamoglobin affinity for oxygen is different in lungs compared to the tissue (see graph - above 66% different)
- Whereas, myoglobin affinity for oxygen compared to the tissue only has 7% difference
- Therefore, heamoglobin changing it’s affinity in different tissues is very important. High affinity in lungs, low affinity for oxygen in tissue
Regulators of oxygen binding - allosteric effectors
Effect of 2,3-bisphosphoglycerate
(can regulate haemoglobin binding to oxygen)
- 2,3-bisphosphoglycerate (found in RBC)
BPG lowers the affinity of Hb for oxygen
• Stabilises the T state (shifts the curve towards the right, lowers the affinity)
How does 2-BPG work to stabilise the T state form?
2,3-BPG is negatively charged: 2,3-BPG interacts with positively charged residues on each b subunit and holds the two beta-subunit together
Regulators of oxygen bidning - allosteric effects
CO2 and H+
CO2 and H+ - the Bohr effect
Binding of CO2 and H+ lowers the affinity of Hb for oxygen
• Stabilises the T state (more in T state)
Allows delivery of oxygen to metabolically active tissues that produce CO2 and H+
Affinity of Hb for oxygen has decreased, it is more likely to release it’s oxygen and less likely to remove it from this location
Bohr shift - look at pic attached (this is where the curve shifts to the right, showing lower affinity for oxygen)
Regulators of oxygen binding - Carbon monoxide
CO binds really tightly to haemoglobin and 250x more readily than O2
- Blocks further oxygen binding once bound
- Stabilises in R state in unaffected subunits– prevents dissociation at tissues (the unaffected subunits therefore have a really high affinity for oxygen that is already bound, it stops this oxygen from dissociating - shifts graph to the left)
Sickle cell disease - what is the mutation, the amino acid properties, effect on the heamoglobin)
- Mutation of Glutamate to Valine in β globin (HbS)
-Val-His-Leu-Thr-Pro-Glu-Lys-
-Val-His-Leu-Thr-Pro-Val-Lys- - Glu lies on the surface of the molecule, in its deoxygenate state, it interacts well with water as GLU IS NEGATIVELY CHARGED
- With the mutation, Val residue then lies on surface in T state (deoxygenated molecule)
4, VAL IS NEUTRAL AND HYDROPHOBIC. Therefore, the heamolgobin has to reorientate itself so the Val is not on the surface near water molecules. Other aa residues from another β -subunits interact with Val. These other aa are hydrophillic. This causes the heamoglobin ON THE SAME RBC to ‘stick together’. This causes a change of shape of the RBC the heam is located on (becomes sickle) - Sickle cells are:
- more prone to lyse (anaemia)
- more rigid, not as flexible (block microvasculature)
What is fetal heamoglobin required?
HbF has a higher binding affinity for O2 than HbA which allows transfer of O2 to foetal blood supply from the mother.
In fetal development, the HbF is much more expressed - look at graph on right
Graph on left shows why oxygen can be transported from maternal oxyheamoglobin to fetel deoxyhemoglobin
Protein targetting - how are cytoplasmic proteins and organelle proteins made?
- Protein destined for cytosol (cytoplasm), or posttranslational import into organelles
- Synthesised on free ribosomes
Protein targetting - How are secretory proteins or membrane proteins made?
- Protein destined for membrane or secretory pathway via co-translational insertion
- Synthesised by ribosomes on the rough ER
An example of secretory proteins being released
Secretory granules - contrinue packaged proteins ready to be released
Can see lots of RER in diagram (as making protein via the ribosome in the RER)
How does the secretory protein get into the endoplasmic reticulum? -> Translation of secreted proteins
- Protein is initially made on free ribosomes
- After the signal sequence (pink) has been translated, it is recognised by the SRP (darker green molecule)
- SRP will bind strongly to signal sequence. It’s binding stops further translation
- The SRP will then bind to the SRP receptor (light darker) (‘docking’ it on the ER)
- Translocon (blue) is a channel associated with SRP receptor
- Hydrolysis of GTP making GDP + Pi drives the opening of translocon channel, as SRP unbinds, this allows translation to start again
- Newly formed protein starts to be extruded into the lumen of the ER
- Signal sequence is chopped by the signal peptide (as it is no longer needed)
- Rest of protein is produced
- Then protein folds
Functions of the Golgi apparatus - Reasons why secretory proteins enter golgi apparatus
- Movement of proteins to the cis-Golgi through budding
- Further protein modifications e.g. further glcosylation
- Release through trans-Golgi to membrane/organelles
Synthetis and modification of collagen in ER
- Synthesis and entry of chain into lumen of rough ER (THIS MADES PREPROCOLLAGEN)
- Cleavage of signal peptide (THIS MAKES PROCOLLAGEN - as the signal sequence is removed)
Procollagen alpha chains are now able to undergo further modications - Hydroxylation of selected proline and lysine residues (leads to the addition of OH groups, catalysed by propyl hydroxylase)
- Addition of N-linked oligosaccharides
- Addition of galactose to hydroxylsine residues
Graph showing the affect of temperature on the helix of: abnormal collagen containing no hydroxyproline and curve showing normal collagen
red curve - as less hydrogen bonding, so triple helix is a lot less stable
Synthesis and modification of collagen in the ER CONT.
What then happens? (2 extra steps)
Prolyl hydroxylase is also involved in the formation of disulphide bonds:
- Chain alignment, formation of disulphide bonds
- Formation of triple-helical pro-collagen from C- to N- terminus (NOTE - 150 extra N-terminal amino acids do no form triple helix, 250 extra C-terminal amino acids do not form triple helix - these are lost later)
Synthesis and modification of collagen in the Golgi
- Completion of O-linked oligosccharide chains by addition glucose
- Transport vesicle as PROCOLLAGEN
Procollagen secretion (from golgi apparatus)
- Exocytosis
Conversion of Procollagen to Tropocollagen
- Removal of N- and C- terminal propeptides
Enzyme: Procollagen peptidase
The role of the C-terminal and N-terminal molecules are to prevent the collagen molecules coming together. Stoping them coming together = stops bundles forming
Formation of collagen fibres (last steps!) (also enzyme required…)
- Removal of the N-termminal and C-terminl propeptides (as described)
- Lateral association of collagen molecules followed by covalent cross-linking
- Aggregates of fibrils (forms collagen fibres)
Lysyl oxidase forms covalent bonds between lysine residues
Checkpoint - What do I need to be able to do?
• An outline of the key features needed for protein secretion
• Differences between constitutive and regulated secretion
• Outline of the structure of collagen
- Triple helical
- Common motifs
- Why the molecule is structurally very stable
• Outline of the processing of collagen
- Where chemical modifications occur
- Where proteolytic cleavage of the molecule occurs
- Names of the different intermediates
