Enzymes regulation, blood clotting, O2 transporters & protein secretion Flashcards
Name 5 short term regulatory mechanisms of enzymes
1) different enzyme forms (isoenzymes)
2) change in enzyme conformation (allosteric reg.)
3) reversible covalent modification (phosphorylation)
4) proteolytic activation
5) controlling amount of enzyme present (gene expression)
What are isoenzymes?
- enzymes that catalyse the same reaction but have different amino acid sequences
- different activity & diff regulatory properties
- synthesised from diff genes or differential spliced from same gene
Give properties of an allosterically regulated protein.
- usually multi subunit
- can exist in 2 forms: T state (low affinity), R state (high affinity)-hint higheR
- do NOT obey Michaelis-Menten kinetics
What do allosteric a) activators AND b) inhibitors do to enzymes in terms of T & R states?
- Activators increase proportion of enzymes in the R state (high affinity)
- Inhibitors increase proportion of enzymes in the T state (lower affinity)
How do the following work?
- protein kinases
- protein phosphotases
- protein kinases add a phosphate group (taken from ATP)
- protein phosphotases remove phosphoryl groups from proteins via hydrolysis (reverse effects of kinases)
Why is protein phosphorylation so effective?
- free energy of Pi bond in ATP is very high
- adds two negative charges (PO4^2-)
- phosphoryl group can make H bonds
- rate of (de)phosphorylation can be adjusted
- allows for amplification effects
What is proteolytic action as a regulatory mechanism for enzymes?
-inactive precursor molecules (zymogens or proenzymes)
-breaking of peptide bond
-irreversible therefore can be regulated
-important when processes need to be tightly controlled
eg digestive enzymes: trypsinogen (inactive)-> trypsin (active)
Blood clotting is a tightly regulated process. What is meant by amplification?
- blood clotting is a series of reactions catalysed by enzymes
- each step leads to amplification of previous signal via cascade mechanism
- very small amounts of initial signal needed to trigger formation of clot
What happens in the intrinsic and extrinsic pathways for blood clotting? At which common point do they meet?
- intrinsic= damaged endothelial lining of blood cells promotes binding of factor XII (7)
- extrinsic= trauma releases tissue factor (factor III ie 3)
- both meet in factor X (10) activation
Thrombin is then activated which forms the fibrin clot. How does this happen?
- prothrombin (inactive) activated to thrombin.
- fibrinogen is a precursor for fibrin. Has 2 heads, alpha helixes. Fibrinopeptides prevent fibrinogen molecules coming together.
- thrombin cuts off these fibrinopeptides to form fibrin. Fibrin assembles to form a ‘soft clot’-cross linking by covalent bonds catalysed by Factor XIII (7).
How is the activation of components in the pathway sustained?
- Factors VIII & V are cofactors that stimulate activity of other enzymes in pathway
- thrombin allows positive feedback on factors VIII, V and XIII, XI
What is the role of gamma-carboxyglutamate residues (Gla)?
hint: vitamin K
- post translationally modifies factors II, VIII etc
- addition of COOH to carboxyglutamate to form Gla requires Vitamin K
- allows interaction w damaged sites & brings clotting factors together
How is the clotting process stopped?
1) localisation of (pro)thrombin- dilution of clotting factor ps and removed by liver
2) digestion by proteases- eg Factors Va & Vllla degraded by protein C (protein C activated by thrombin negative feedback loop)
3) binding of specific inhibitors eg antithrombin III
State the similarities & differences between haemoglobin and myoglobin.
- haemoglobin= in blood, transports O2 around body, 4 polypeptide chains, 4 haem groups per Hb
- myoglobin= in muscle, short term storage of O2, 1 polypeptide chain, 1 haem group per Mg
- in both, O2 binding changes position of Fe ion
What is the shape of the slope in O2 binding with a) myoglobin
b) haemoglobin
Why is this so?
- myoglobin= hyperbolic ie myoglobin has a constant affinity for O2
- haemoglobin= sigmoid also (S shape), affinity for O2 increases w partial pressure for O2
- this is because of the structural differences, cooperativity enhances O2 transport in Hb; binding of first O2 is harder but causes conformational change making binding of next O2 easier. It promotes transition from low affinity T state to high affinity R state. This property means Hb has greater O2 transport than Mglbn
Name an allosteric regulator of O2 binding. Describe how it works and what is does.
- BPG (1,2,3-bisphosphoglycerate)
- it lowers the affinity of Hb for O2 therefore stabilises the T state
- it does this by interacting w the positively charged residues on each beta subunit on Hb
- it shifts the curve to the right
Other allosteric regulators of O2 binding are CO2 & H+ ions.
Explain how these work & what is this effect called?
In which direction will the curve shift?
- Bohr effect
- CO2 and H+ are both acidic, binding of these lowers Hb’s affinity for O2 meaning it stabilises the T state
- the curve shifts to the RIGHT
- allows delivery of O2 to metabolically active tissues that produce CO2 & H+
Another regulator of O2 binding is carbon monoxide (CO).
How does it work & what effect does it have in the body?
- importantly, CO binds to Hb 120x more readily than O2
- this means it can block further O2 binding once bound
- it stabilises the R state in UNaffected subunits meaning O2 is stuck on Hb & unable to dissociate at the tissues
Which gene mutation causes sickle cell anaemia?
What are the consequences of this disease?
- mutation of glutamate to valine
- Val lies on Hb surface in T state molecule (deO2)
- sickled cells are more likely to lyse and become rigid meaning they don’t have as large a capacity to carry O2
What is the key property of fetal Hb ?
Why is this useful?
- fetal Hb has a higher affinity for O2 than maternal Hb
- this means any O2 maternal Hb has will be transferred to fetal Hb.
- this is important as it’s the only O2 supply the foetus has
What is protein targeting? How does the cell do this?
- making sure proteins go to the correct location to work
- proteins destined for cytosol synthesised on free ribosomes
- Proteins destined for membrane or secretory pathway synthesised and ribosomes on RER
What are the two different types of protein secretion?
1) constitutive (ongoing all the time eg albumin production in blood)
2) regulated eg endocrine cell (hormones), exocrine cells (digestive enzymes), neurocrine cells (NTs)
What is required for protein sorting?
- a Signal intrinsic to the protein
- A receptor that recognises the signal and directs it to the correct membrane
- A translocation machinery
- Energy to transfer the protein to a new place
What is a signal sequence?
-give an example
- eg preproalbumin
- pre part is the signal sequence that is removed during processing
- N terminal amino acid sequence
- 5 to 30 aa’s
- able to form alpha helix
- central region rich in hydrophobic residues
The endoplasmic reticulum is responsible for some post translational modification. Give some more of its functions
-insertion of proteins into membranes
-specific proteolytic cleavage -glycosylation
-formation of S-S bonds
proper folding of proteins
-hydroxylation
-assembly of multisubunit proteins
Give some of the functions of the Golgi apparatus
- Movement of proteins to the Cis-golgi through budding
- further protein modifications
- release through Trans-golgi through to membranes or organelles
Collagen is an example of a secreted protein. where does synthesis and modification of collagen take place?
-the endoplasmic reticulum
What is the basic unit of collagen? Give some of its properties and structure
- tropocollagen
- 300nm rod shaped protein
- 3 polypeptide chains
- characteristic triple helix (right handed)
- H bonds between chains stabilises it
Outline the process of the synthesis and modification of collagen.
Remember CHADPOGRL
C-cleavage of signal peptidases
H-hydroxylation of proline and lysine (in scurvy no VitC means this can’t happen)
A-addition of N linked oligosaccharides and galactose in ER
D-disulphide bridge formation
P-procollagen transported to Golgi
O-o linked glycosylation in Golgi
G-golgi ; exocytosis as tropocollagen
R-removal of N/C terminal peptides using pro collagen peptidase
L-lateral aggregation to form fibrils
