Dr Leylands Bit Flashcards
Which isomer of amino acids is found in proteins?
L (left handed) isomer
List eight functions of proteins.
Transporters Catalysts - enzymes Structural support - collagen Ion channels Machines - muscle contraction Immune protection Receptors Ligands in cell signalling.
What are the non polar amino acids?
Glycine, alanine, methionine, leucine, isoleucine, proline, phenylalanine, tryptophan, valine.
What are the charged amino acids?
Lysine, arginine, histidine, aspartate, glutamate
What are the polar uncharged amino acids?
Serine, threonine, asparagine, glutamine, tyrosine, cysteine.
Which are the aromatic amino acids?
Phenylalanine, tryptophan
What is KR, what would you expect the value to be for a stronger acid?
KR is the acid dissociation constant for the R group, represents the proportion of molecules that dissociate in a solution.
It is higher for a stronger acid.
What is pKR, what value would you expect for a stronger acid?
pKR is -log(KR) (log to base ten!!!!)
Lower for a stronger acid.
If the pH of a solution is less than pKR, would you expect the group to be protonated?
Yes.
If the pH of a solution is greater than pKR, would you expect the R group to be protonated?
No.
What are the main organelles in a cell, and their functions?
Plasma membrane - controls what enters and leaves the cell
Smooth endoplasmic reticulum - synthesis of lipids
Vacuole - storage of water and waste products
Golgi apparatus - modifies proteins from RER and secretes them.
Rough endoplasmic reticulum - post translational modification of proteins
Nucleus - contains DNA
Lysosomes - contain hydrolytic enzymes for intracellular digestion
Basal bodies - base structures of cilia and flagella.
Mitochondria - synthesis of ATP
Ribosomes - translation of mRNA to protein.
Cytosol - contains soluble enzymes, substrate for diffusion.
What bonds are important in macromolecular structure and interaction?
Hydrogen bonds
Hydrophobic interactions
Ionic interactions
Van der waals interactions
What are the properties of a peptide bond?
The N-C peptide bond has partial double bond characteristics, so it is rigid and planar. The alpha carbons, carboxylic acid carbon, the nitrogen and the hydrogen and oxygen all lie in a plane.
They are always trans, not cis.
What is the pI?
The isoelectric point of the protein - the pH at which is has no net charge.
If the pH>pI, what is the net charge on the protein?
Negatively charged as the protein will donate its proton.
If pH<pI would the protein be positively or negatively charged?
Positively as it will accept a proton.
Would an acidic protein have a high or low pI?
Low
Would a basic protein have a high or low pI?
High
How many residues per turn of an alpha helix?
3.6
What is the pitch of an alpha helix?
0.54nm
Are alpha helices right or left handed?
Right handed.
Between which atoms do hydrogen bonds form in an alpha helix?
The oxygen from the carboxy group and the hydrogen from the amino group four amino acids away.
What are the two main secondary structures of proteins?
Alpha helices and beta sheets
Which residues are strong helix formers and why?
Alanine and leucine.
They are small and hydrophobic.
What residues are helix breakers?
Proline, because you can’t rotate around the bond between the alpha carbon and the nitrogen.
Glycine because it has H as its R group which supports other conformations - rotation is unconstrained.
Consider a beta strand. What is the distance between adjacent amino acids, and what is the configuration of R groups?
0.35nm
R groups alternate between either side of the chain.
What are the different arrangements of beta strands to make a beta sheet?
Parallel - adjacent strands run in the same direction, stabilised by hydrogen bonds.
Antiparallel - adjacent strands run in opposite directions, stabilised by hydrogen bonds.
Mixed
Give two examples of motifs in a tertiary structure.
Beta - alpha - beta loop
Beta barrel
What is a domain of a polypeptide?
Part of a polypeptide chain that folds into a specific shape and often has a specific function.
If a polypeptide was folded such that hydrophobic side chains are buried inside the polypeptide and hydrophilic side chains are exposed, what could you conclude about the polypeptide?
It is likely to be water soluble.
How would you expect hydrophobic and hydrophilic residues to be arranged in a membrane protein?
You would expect hydrophilic residues in the centre lining a hydrophilic channel, and a hydrophobic exterior that will be embedded in the plasma membrane.
What forces are involved in maintaining the primary structure of a protein?
Covalent bonds.
What forces are involved in maintaining the secondary structure of a protein?
Hydrogen bonds.
What forces are involved in maintaining the tertiary and quaternary structures of a protein?
Covalent (disulphide) bonds, hydrophobic interactions, ionic bonds, van der waals forces, hydrogen bonds, electrostatic interactions between charged groups (salt bridges)
What residues are disulphide bonds formed between?
Cysteine
What is the bond energy of a disulphide bond?
214 kJ/mol
What can be used to reduce disulphide bonds?
Beta-mercaptoethanol
What is the usual fate of proteins with disulphide bonds?
Secretion from the cell.
What can denature proteins and how.
Heat increases vibrational energy, disrupts intermolecular forces
pH alters ionisation states of residues, alters ionic and hydrogen bonds.
Detergents and organic solvents disrupt hydrophobic interactions.
What diseases promote misfolding of proteins?
Transmissible spongiform encephalopathies
What are amyloidoses?
They are accumulations of a misfolded, insoluble form of a normally soluble protein. There is a high proportion of beta sheets which form before the rest of the protein.
It is stabilised by hydrophobic interactions between aromatic amino acids.
What diseases are characterised by amyloidoses?
Alzheimer's disease Parkinson's CJD Huntingdons Type 2 diabetes mellitus
What is the physiological role of myoglobin?
Myoglobin is an oxygen carrier in muscle, and also acts as a reservoir for oxygen in muscle.
What is the physiological role of haemoglobin?
Found in erythrocytes, transports oxygen from lungs to capillaries, and carbon dioxide and hydrogen ions from the capillaries to the lungs.
What is the structure of haem, and what is its function?
Porphyrin ring, with a central iron atom bound to the 4 nitrogen of the ring.
It binds oxygen. The Fe2+ iron can bind with two oxygen atoms, one on each side of the plane.
How is haem bound to the protein in haemoglobin and myoglobin?
By the proximal histidine residue, which binds the Fe atom.
Describe the structure of myoglobin
153 amino acids
75% alpha helical
Compact, globular
His 93 in the 8th alpha helix is covalently bound to iron.
How does oxygen binding alter the conformation of haem?
When oxygen binds the Fe atom it moves the Fe into the plane of the porphyrin ring, produces conformational change in the protein.
How does oxygen binding depend on oxygen saturation (oxygen dissociation curve) for haemoglobin and myoglobin?
Myoglobin: hyperbolic
Haemoglobin: sigmoidal.
What states can haemoglobin exist in, and which is favoured by oxygen?
T state (low affinity for oxygen) and R state (high affinity for oxygen). Oxygen binding favours R state.
What is the advantage of sigmoidal binding curve of haemoglobin?
Sensitive to small changes in oxygen concentration, efficient transport of oxygen from lungs to tissues.
What is the effect of an increase in the concentration of 2,3 bisphosphoglycerate on the affinity of haemoglobin for oxygen?
Reduces the affinity of haemoglobin for oxygen
Where does 2,3 BPG bind?
The centre of the haemoglobin tetramer.
What increases 2,3 BPG concentration?
High altitude.
What is the Bohr effect and why is it physiologically important?
The affinity of haemoglobin for oxygen is decreased by hydrogen ions and carbon dioxide.
Metabolically active tissues produce large amounts of hydrogen ions and carbon dioxide. Couples delivery of oxygen with demand.
Why is CO poisoning fatal, and percentage saturation is fatal?
CO binds to haemoglobin with 250x higher affinity than oxygen and increases the affinity of the other subunits for oxygen (meaning less oxygen dissociates in the tissues)
This blocks oxygen transport. More than 50% saturation is fatal.
What are the three main types of haemoglobin in an adult, and in what proportion are they present?
HbA (two alpha and two beta chains) 90%
HbF (two alpha and two gamma chains)2%
HbA2 (two alpha and two delta chains) 2-5%
How does the affinity of foetal haemoglobin for oxygen differ from adult haemoglobin, and why is this important?
Foetal haemoglobin has a higher affinity for oxygen which allows transfer of oxygen from maternal blood supply to the foetal blood supply.
What is the mutation in sickle cell anaemia?
Glutamate to valine
What is the consequence of the mutation in sickle cell anaemia?
Creates a sticky hydrophobic pocket, which allows the HbS to polymerise.
What is the consequence of having sickled red blood cells, and what symptoms does this cause?
The cells are more prone to lyse - anaemia
More rigid, block capillaries - sickle cell crisis
What is a thalassaemia?
Genetic disorder causing imbalance between the number of alpha and beta chains in haemoglobin
What is beta thalassaemia?
Decreased or absent beta globin chain production
Alpha chains can’t form stable tetramers
Appears after birth as in a foetus tetramers form with gamma subunits (HbF)
What is an alpha thalassaemia?
Decreased or absent production of alpha globin subunits
Multiple levels of severity as two adjacent genes code for alpha globin.
Present before birth
Beta chains form stable tetramers with increased affinity for oxygen.
How do enzymes catalyse a reaction?
Facilitate formation of transition state, lower activation energy.
Name six important features of enzymes
Highly specific May need cofactors Unchanged by the reaction Don't change the equilibrium Proteins Increase rate of reaction
Explain the induced fit hypothesis
Binding of substrates changes conformation of the enzyme, promotes formation of transition state.
Substrate binds enzyme by non covalent bonds.
What does the Michaelis Menten model predict, and what is the equation? Do all enzymes obey this model?
That an enzyme substrate complex is a necessary intermediate in catalysis.
V0=Vmaxx[S]/(km+[S])
No
Define Vmax
Maximal rate of reaction when all enzyme active sites are saturated with substrate.
Define Km
The concentration of substrate that gives a rate of reaction equal to half of Vmax
What are the Km values of hexokinase and glucokinase, and what is the significance of this?
The km of hexokinase is 0.1mM, implies a high rate of reaction and high affinity for the substrate at low concentrations so glycolysis occurs even when glucose levels are low.
The km of glucokinase is 5mM, it is the present liver and helps reduce the concentration of glucose when it peaks after a meal, implies a low affinity for glucose.
What is the lineweaver-burk plot, and what is the advantage of it?
Rearrangement of Michaelis Menten equation to give a straight line, allows for easy estimation of Vmax and km
Plot 1/V0 against 1/[S]
1/V0=(km/Vmax)*1/[S]+1/Vmax
Give an example of an irreversible inhibitor of an enzyme.
Nerve gases such as sarin - form covalent bonds
What is the effect on km and Vmax of a competitive inhibitor?
Km will be higher, Vmax will be the same.
What’s the affect on km and Vmax of a non competitive inhibitor?
Doesn’t affect km, Vmax is lower.
How can enzymes be regulated?
Alter rate of synthesis Alter rate of degradation - ubiquituin Change substrate and product concentration Proteolytic cleavage Covalent modification - phosphorylation Allosteric regulation
What are isoenzymes?
Different forms of an enzyme for example hexokinase and glucokinase that have different kinetic properties.
What is product inhibition?
Accumulation of product inhibits forward reaction - for example glucose 6-phosphate inhibits hexokinase
What is the relationship between rate and substrate concentration in an allosteric enzyme?
Sigmoidal
What states do allosteric enzymes exist in?
T state - no substrate bound, low affinity
R state - high affinity
What is the effect of allosteric inhibitors and activators?
Bind at a site other than the active site
inhibitors increase the proportion of enzymes in the T state
Activators increase the proportion of enzymes in the R state.
What can you say about the number of active sites on an allosteric enzyme?
Must have more than one!
What reaction does phosphofructokinase catalyse?
Conversion of fructose 6-phosphate to fructose 1,6-bisphosphate.
This is the rate limiting step of glycolysis.
How is phosphofructokinase regulated allosterically?
Activated by AMP, fructose 2,6 bisphosphate
Inhibited by ATP, citrate, H+
Give an example of an enzyme regulated by phosphorylation
Glycogen phosphorylase - activated by phosphorylation.
Pi donated from ATP
Where is the phosphate transferred to and from in phosphorylation?
From terminal phosphate of ATP to the OH group of serine, threonine or tyrosine.
What catalyses the phosphorylation and dephosphorylation of proteins?
Protein kinases and protein phosphatases
How does the number of activated enzymes increase in an enzyme cascade?
Geometrically, which can lead to an increase of several orders of magnitude in a few milliseconds.
How are glycogen breakdown and synthesis regulated?
Reciprocally.
Adrenaline activates adenylate cyclase
Adenylate cyclase converts ATP to cyclic AMP
Cyclic AMP activates protein kinase A.
This phosphorylates glycogen synthase, inhibiting glycogen synthesis.
And it phosphorylates phosphorylase kinase, which phosphorylates glycogen phosphorylase, stimulating glycogen breakdown.
What is a zymogen?
Inactive precursor of an enzyme
Where is pepsinogen synthesised, and what is it cleaved to form?
Stomach, pepsin
Where is Chymotrypsinogen synthesised and what is it cleaved to?
Pancreas, Chymotrypsin.
What is Trypsinogen cleaved to, and where is it synthesised?
Trypsin, pancreas.
What is procarboxypeptidase cleaved to and where is it synthesised?
Pancreas, carboxypeptidase.
What is proelastase cleaved to and where is it synthesised?
Elastase, pancreas
How is apoptosis mediated?
Mediated by caspases, synthesised in zymogen form as procaspases
How is chymotrypsinogen activated?
Proteolytically cleaved by trypsin into pi chymotrypsin (active)
It is then further cleaved (autoactivation) to form alpha chymotrypsin (active) - consists of three peptide chains joined by disulphide bonds.
How is trypsinogen activated?
Enteropeptidase (lining pancreatic duct) cleaves trypsinogen to trypsin.
What is the role of trypsin?
Controls the activation of pancreatic proteases, chymotrypsin, lipase, elastase and carboxypeptidase.
What causes emphysema?
Deficiency of alpha-1 anti trypsin
Alpha-1 anti trypsin is an endogenous inhibitor of trypsin, a deficiency leads to a destruction of alveolar walls by elastase, that isn’t activated if there is sufficient alpha-1 anti trypsin that prevents trypsin from cleaving proelastase.
What is alpha-1 anti trypsin?
Inhibits proteases
53kDa
Plasma protein.
What is the first point common to both extrinsic and intrinsic clotting pathways?
Activation of factor 10
How does the intrinsic pathway lead to the activation of factor 10?
Factor 11 (endopeptidase - thromboplastin antecedent) activates factor 9 (endopeptidase with gla residues - christmas factor) Factor 9 activates factor 10 (endopeptidase with gla residues - Stuart Prower factor). Requires factor 8 as a cofactor (antihemophilia factor b)
What activates the intrinsic pathway?
Membrane damage causes aggregation of platelets. Calcium ions bind to the phospholipid bilayer of platelets.
Factors 9 and 10 undergo post translational modification in the liver - carboxylic acid groups are added to glutamate residues - forms gamma carboxyglutamate residues (gla). These are negatively charged so bind to the calcium,
By what means does the extrinsic pathway activate factor 10?
Damage to tissues exposes tissue factor (factor 3).
Causes autocatalytic activation of factor 7 to 7a
Activates factor 10 to 10a
What happens following the activation of factor 10 to 10a?
Activates prothrombin to thrombin (requires factor 5)
Activates fibrinogen to fibrin
Converted to cross linked fibrin (by factor 13, activated by thrombin)
What is the structure of prothrombin
Protease part is at the C terminal.
Gla domains at the N terminal target it for activation
Two Kringle domains maintain in the inactive form
How is prothrombin cleaved?
Cleaved at Arg274 releases fragment containing Gla and two Kringle domains.
Cleaved at Arg323 - fully active thrombin - 2 chains 6kDa and 31kDa linked by disulphide bond.
WhAt ensures only prothrombin at the site of damage are activated?
It binds calcium ions which accumulate at sites of membrane damage via Gla residues.
WHat is the structure of fibrinogen?
340kDa
2 sets of 3 polypeptides (alpha, beta, gamma) held together at N terminus by disulphide bonds.
N terminals negatively charges - prevents aggregation
3 globular domains
How does a soft fibrin clot form?
Thrombin cleaves fibrinopeptides from central globular domain of fibrin
Globular domains at C terminal of beta and gamma chains interact with exposed sequences at N terminal of alpha and beta chains forming a fibrin mesh
What causes a soft clot to be stabilised?
Transglutaminase (factor 13) is activated by thrombin.
Catalyses the formation of peptide bonds between lysine and glutamine.
What is the consequence of a defect in factor 8, and how is it treated?
Classic haemophilia - factor 8 stimulates the activity of factor 9, and it is involved in positive feedback and amplifying the activity of the cascade in response to proteolysis by thrombin.
Treated with recombinant factor 8.
What does thrombin proteolytically activate?
Fibrinogen Factor 13 (transglutaminase) Factor 8 Factor 11 Factor 5
What is the role of vitamin K in blood clotting?
Vitamin K is involved in the gamma carboxylation of glutamate residues on factors 2, 7, 9 and 10
How does warfarin anticoagulate?
Inhibits vitamin K epoxide reductase which recycles oxidised vitamin K which has been involved in carboxylation of clotting factors, reducing the availability of vitamin K as a cofactor.
What stops the clotting process?
Dilution of clotting factors by blood flow and disposal by the liver
Protein C is activated by thrombin bound to thrombomodulin (Transmembrane protein), and EPCR (endothelial protein C receptor) which is bound to the endothelium. Protein C degrades factor 5 and 8.
What is the consequence of protein c deficiency?
Thrombotic disease
What is antithrombin 3?
Plasma protein that inhibits the activity of factor 9, 10, 11, 12, 2(thrombin) and 7 when activated.
Enhanced by heparin.
Does not act on thrombomodulin bound thrombin (which activates protein c)
What does streptokinase do?
Activates plasminogen to plasmin, breaks down fibrin clots into fragments.
Used in MI
What activates plasminogen?
Streptokinase. t-PA (tissue plasminogen activator)
