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Flashcards

Enzymes

Jenna Robinson Biology English 36 cards

Cards in this deck (36)

  1. 1Describe activation kinetics and why it is seen- Limited number of active sites - if substrate conc is high enough, catalyst becomes saturated
    - At high substrate concentrations V=kcat[E]0 - so it tells you about the overall efficiency of the aka the turn over number - number of products produced per active site per enzyme for saturating substrate levels - also tells you about the free energy difference between the ES complex and the transition state
    - Km is a measure of binding - generally for all enzyme bound species but particularly for the substrate - this is true if kcat<
  2. 2Where do enzymes bind most strongly and what implications does this have for synthesising inhibitors?- Stabilisation of transition state - must bind to the transition state more than the substrate of the complex
    - If a reaction involves the accumulation of charge this can be costly and enzymes can reduce this energy
    -inhibitors are designed to mimic transition states - e.g. phosphoamidate is an inhibitor of acyl transferases - it is tetrhedral - replaces the carbonyl group at which the peptide cleavage occurs - binds more strongly to the enzyme the the substrate - means it can out-compete it even in low concentrations
  3. 3How does the entropic effect help with enzyme catalysis- Entropic effect - bring substrates together on the enzyme - removing their individual degrees of freedom
    - Intramolecular reactions are much faster than intermolecular reactions - this can be measured by the effective molarity which is the hypothetical concentration of the second reactant that would be required to observe the rate constant seen by intramolecular reaction - can vary from 10 ^2-10^8 M - surprising as concentration of water is only 55M this is because reaction would happen via an encounter complex - but the colliding molecules would retain most of their freedom to move - in an enzyme the freedom is much more restricted
    -Enzymes can make reactions pseudo-intramolecular by binding to substrate which then reacts intramolecularly with the active sites in the enzymes
    -This is favourable as it reduces the overall entropy loss of reaction as it is compensated for by the energy of binding to the enzyme - the size of this effect is enough to account for almost all of enzymes rate enhancement of up to 10^8
  4. 4What is nucleophilic catalysis and how does it increase rate of reaction?- Often form a covalent bond in catalysis
    - E.g. in peptide hydrolysis by chymotrypsin - serine attacks first - makes the amide a more reactive ester - better than direct attack by h2o as alcohols are better nucleophiles, and the covalent linkage to the substrates gives it a more rigid handle - stops it diffusing off
  5. 5Describe general acid and general base catalysis- RNH- is a poor leaving group: pKa ~35 - need to make the leaving group better, this can be by acid catalysis - transfer of proton in transition state - lowers its energy
    - If a relatively unreactive nucleophile e.g. water or serine (pKa ~14) is used - may be better to deprotonate using a base
  6. 6Describe two that metal ions aid with catalysis1. increases reactivity of nucleophiles - e.g. metal ions coordinated with water reduces the pKa so much that a significant concentration of the reactive -OH group is present e.g. pKa of cobalt bound water is ~6.6
    2. Also helps stabilisation of -ve charge on transition state - e.g. Lewis acids in leaving group stabilisation - sometimes they are called 'superprotons' because they have the same effect but are pH independent
  7. 7What does the hydrophobic effect have on catalysis?-Enzyme active sites can be very hydrophobic - means the pKas of residues can change a lot - carboxylic acids will go up, amine bases will go down - as charge is destabilised
    - H phobic interactions will also reinforce charge-charge interactions compared to in aqueous solution
    - The may also be desolvated -which increases its activity -same effect is seen when rate accelerations increase in organic solvents
  8. 8Describe the features of a-chymotrypsin- Protelotic enzyme - part of the serine proteases - as they all contain an active-centre serine directly involved in catalysis
    -Examples include chymotrypsin and and trypsin - released from the pancreas as zymogen, elastase - degrades elastin, thrombin - involved in blood clotting, plasma - dissolves blood clots
    - chymotrypsin is typical as it is synthesised as an inactive precursor - chymotypsoinogen which is activated by cleavage by tyrpsin at agr15/Ile 16
    -Further action by chymotyrpsin itself leads to excision of 2 dipeptides - cut at ser14-arg15 and Thr147-Asn158 - to give the a-chymotrypsin which is normally studies
  9. 9How were the kinetics of the enzyme characterised?- You challenge it with a variety of substrates and then measure Michelis menten kinetics
    - By using synthetic substrates it showed that there was a preference for large, aromatic amino acids
    - Hartley added an unusually large amount of enzyme to 4-nitrophenylacetate and saw a burst of '4-nito-phenol' - suggests an accumulation of intermediate and biphasic kinetics i.e. first step (substitution by serine195) is quick because there is a good leaving group, second step is slow
    - See book for pic on burst kinetics
  10. 10How can fast kinetics be measured?- Can be measured using stopped flow instrument - enzyme and substrate are rapidly mixed to together and then stopped by opposing flow and the reaction progress is monitored
    - This type of kinetics aka 'pre-steady' or single-turnover kinetics because it has before the equilibria is et up that are the basis for Michaelis-menten e.g. that the concentration of E.S remains the same
    - This approach is important when the existence of intermediates and their rate formation and breakdown are of interest
    -but we must be careful not to over interpret biphasic burst kinetics - can be seen when there is strong product inhibition i.e. when the product binds more strongly to the enzyme than the substrate - blocks binding after first turnover
  11. 11How can covalent chemical modification be used to study enzymes?- Chemically modify a residue in the active site - if it results in reduced catalytic activity could assume its involved in catalysis
    - But you must check that you're specifically targeting a residue - i.e. must characterise the modified protein
    - Even then, the size of the modification may just disturb or block the active site even if it is not essential for the mechanism
  12. 12How was covalent modification applied to chymotrypsin?- Modification with TPCK - inhibitor that mimics substrate as it has a aromatic ring which directs to active site (of chymotypsin normally cuts at L-Phe residue) - the reactive chloro ketene group then reacts with any nucleophile available - but labelling was not on serine but his57
    -DIFP is an oxygen specific reagent- specifically labels Ser 195 - although there are nearly 30 other serine in the enzymes - being protonated at physiological pH, serine is not normally nucleophilic, but this particular serine is due to its position in the active site - after modification the protein was hydrolysed and chromotographic analysis of protein
    -But this only shows which residues are in the active site, not necessarily how much they contribute to catalysis
  13. 13What has crystal structure revealed about the catalytic triad in serine proteases- His, ser and also aspartate102 which wasn't identified by chemical modicfication - revealed a charge relay system where the position of the serine helps it be a better nucleophile
    - Additonally NH groups from the backbone create and oxyanion hole which doesn't contribute much to the stability of enzyme-substrate complex, but does lower the energy of the transition state which has a negative charge
  14. 14How can protein engineering be used to probe for catalytic residues?- Alanine scanning e.g. subtilisin is a serine protease where the catalytic triad has been replaced with alanine
    - E.g. ser195ala and his57 ala mutants have cat 10^6 times lower - but Km is unaffected - showng they are important for catalysis
    - If ser/his/asp ---> ala/ala/ala the rate acceleration is still 10^4 fold, showing that catalytic contribution on the oxyanion hole
  15. 15Where is the catalytic triad seen beyond the serine proteases?- Carboxypeptidase A
    - The general base is no a carboxylic acid and not histidine
    - The nucleophile (water) is also coordinated to a metal ion - this increases its reactivity but also removes the problem of binding and positioning water in the enzyme as it is such a small molecule - additionally allows for fine tuning, as reaction doesn't just depend on presence of the metal ion but on the geometry of the other ligands
  16. 16How is the coagulation cascade initiated, regulated and inhibited?- Uses serine proteases known as coagulation factors - important to prevent bglood loss after injury
    -Organisation into a cascade allows for amplification as well as multiple opportunities for regulation
    - The platelet surfaces eventually acts as a template for the assembly of tenase complex which converts factor X to factor Xa which converts prothrombin to thrombin - which clips two short peptides from fibrinogen to produce fibrin which spontaneously form fibres that interact with platelets - stabilising the clot and trapping red blood cells in a haemostatic plug
    - Pathway is diverted by blood-sucking parasites like ticks as they have salivary protein which binds a hydrophobic cleft adjacent to the active site of thrombin - displaces the catalytic triad
  17. 17What can pH rate profiles tells us about reactions- See books for pics
    - Charge may result in inhibitory effects of the enzyme
    - At high pH the b-carboxylate group of aspartate-194 block the specificity of the pocket of chymotrypsin, at neutral pH, the protonated isoleucine amino group forms an ion pair with the aspartate carboxylate group - opening up the pocket for hydrophobic substrate side chains to bind
  18. 18What evidence is there that the catalytic triad has been conserved?- Often amino acid sequence is very different between serine proteases but the 3D structure is identical - same one fold and one arrangement of residue in the active site
    - It is even possible to implement that the catalytic triad is an entirely different fold e.g. subtilisin in bacteria, has different secondary structure, overall fold , no sequence homology and a different of critical polypeptide chains - yet the positions of the critical groups in the 3D structure are identical - convergent evolution
    - Good because once you understand the mechanism in one organism, you understand it in all of them
  19. 19What have comparative studies told us about the structure of the binding pocket?- Comparison on trypsin and elastase shows that there specificity is due to hydrophobic residue directly to the n-terminal of the bond being hydrolysed - the binding pocket is lined with hydrophobic side chains
    - When chymotypsin is activated, further changes occur - when chymotypsinogen is activated by limited proteolysis- conformational change means there is a new salt bridge between asp194 and Ile16 - this causes the substrate-binding pocket to form by movement in residues 189-192
  20. 20What does protein engineering tell us about the details of serine proteases?- Alteration of specificity was attempted by increasing the bulk of both side chains in the specificity pocket - normally glucose - change to alanine - this disfavoured the binding of arginine, but increased binding of lysine
    - Replacement of asp102 for asn which has the same shape but a different charge was used to deduce its role - as it is inaccessible to chemical modification - the mutant rat tyrpsin was expressed at high levels by establishing a stable eukaryotic cell line that secreted tyrpsinogen into the medium - wild type enzyme purified from the cell line was identical to the normal rat enzyme, but the mutant had a kcat 2x 10^-4 of the normal - important for catalysis, but the km was only twice as large - not as big as an effect
    - X-ray structure revealed the asparagine acts as an H-bond donor to His57, whereas Asp102 acts as a acceptor
  21. 21With reference to aspartate transcarbamoylase (ATCase) describe how allosteric regulation can used to control enzyme- Catalyses the synthesis of pyrimidines de novo - transfer of a carbamoylase to aspartic acid - carbamoylase phosphate reacts with aspartic acid to form pi and n-carbamoyl aspartate - then goes on to produce CTP and pyrimidines
    - At fixed concentration of second substrate, showed a sigmoidal curve when increases the start substrate - cooperatively - switch over a narrow concentration
    -CTP inhibits ATCase - forward inhibition - but can be outcompeted by excess aspartic acid - never reaches 100% - so does not follow Michaelis menten
    - ATP is an activator - thought to bind to the same site on ATCase as CTP as CTP inhibition can be removed by excess ATP - it increases affinity but doesn't change V - the biological consequence may be to regulate purine/pyrimidne residue - so mechanism isn't activated wastefully
  22. 22What effect does the addition of mercury containing molecules have on ATCase activity? How can this be studied?-Hg has high affinity for -SH units on cysteine - removes allosteric activity but not catalytic activity - returns to Michaelis menten behaviour
    - ultracentrifugation studies show that Hg blockage causes enzyme to dissociate into 3 regulatory units and 2 catalytic units - breaks disulfide bridge and H bonds -this can be reversed by adding excess thiol e.g mercaptoethanol - the subunits recombine and allosteric regulation returns
    - If the subunits were not held together by disulphide bonds, the sedimentation pattern should be the same before and after - number of peaks = number of subunits - once dissociated you see two peaks
  23. 23Describe the structure of ATCase? How was this structure found- catalytic large subunit - catalysis, no CTP inhibition, c3 - three 34 kDa subunits
    - Regulatory subunits - CTP inhibition - two 17 kDa chains - r2
    -Enzymes = 2xc3 + 3xr2 = c6r6
    - X-ray crystal structure using PALA - a transition state analogue of the enzyme - higher km but same kcat - it binds to all 6 subunits of the active site
    -Allosteric interactions involved an entire change in quaternary structure
  24. 24What has protein engineering showed us about the catalysis in ATCase- Mutation of Lys84 to arginine resulted in a decrease of kcat/km but not km - suggests important for catalysis - suggests stabilisation of transition state but not the substrate itself
    - Mutation of ser80 has not effect on kinetic parameter - irrelevant for catalysis
  25. 25How does the structure of ATCase change on substrate binding?- The binding of L-aspartate decreases the sedimentation coefficient by 3% - suggests swelling of the enzyme (increased friction coefficient, f) - comparison of X-ray structure with PALA bound vs not shows that the subunits move 12 A apart and rotate slightly when substrate is bound
    - Also causes ser80 and lys84 to move to join each active site subunit
    - R and T state - R favoured by substrate binding - more active, T state less active, favoured by CTP biding
    - But this rearrangement isn't caused by one residue - many interactions - complex network - makes engineering de novo very hard
  26. 26What experiments were conducted to show that all subunits interact- hybridisation studies show cross-talk between the two catalytic subunits -if one trimer is inactive and has a chromophoric receptor binding to the other trimer is detected by the reporter group
    -Similar experiments also show cross-talk between the catalytic and regulatory subunits
    - Mix labelled inactivated c3 trimer with native c3 and r2 units and use substrate analogues to activate the active c3 - then purify hybrids - shows that fluorescence changes on binding - so the top trimer is able to 'sense' quaternary shift for the bottom trimer
  27. 27What is the general structure of antibodies and how is it manipulated in the study of proteins- IgG is an immunoglobulin that is Y shaped has 2 light and two heavy chains, variable regions at end of chain -bind antigen and hinge region in middle - multiple s-s bonds, carbohydrate is attached to heavy chain
    - Treatment with protease causes cleavage of mobile region giving rise to two antigen-binding fragments - Fab - the residual constant part if Fc - doesn't have binding
    - Since Fab has binding of entire antibody this is often used to study its properties easier to use small proteins in biological approaches
    - For protein-antigen, biding pocket is flat is extensive, but for small molecules it is more cleft-like
    see book for pics
  28. 28What is the immunoglobulin fold and how was it found- 2 antiparallel B strands joined by a disulphide bond
    - Found by amino acid sequencing and X-ray structure of an IgG produced by human myeloma - which have a single species instead of a complex mix
    - Around 110 amino acids - not only found in immunoglobulins but in T cells receptors and proteins of the major histocompatibility complex
  29. 29How do immunoglobins recognise and bind antigens?- Both heavy and light regions within their variable chains contain 3 hyper variable regions termed the complementary determining regions - they are seen in X-ray structures to form the loops which emerge from the framework set up by the B-immunoglobin fold
    - The variable and constant domains in the light chain are made from 2 different globular units
    - The VH interacts with the VL and CH with CL- this brings the CDR of the heavy and light chains together to form the antigen binding site - so involves both chains are is similar to the way that enzymes stabilise substrate binding - charge neutralisation, van Der Waals and other short-range interactions
  30. 30How have monoclonal antibodies been used to study antibody binding? How has it been used to study binding of lysozyme?- Can obtain homogenous materials for crystallisation e.g. monoclonal antibodies have been raised against hen egg lysozyme - Fab fragments have been purified from 27 clones - one was found to crystallise out well
    - Comparison of the structure of the Fab with the complex with lysozyme showed it recognised a discontinuous epitope
    - There is no evidence for conformational change
    - The contact surface area is large and flat - all 6 CDRs play a part - about 17 amino acids residue in total
    - One of the side chains - glutamine-121 penetrates fairly deeply into the antibody - in a mutant where this is changed to histidine you don't get binding - reflects instability of a buried positive charge
  31. 31Give two examples of when binding of antigen causes conformational change in the antibody. Why might this pose problems?1. Influenza virus enzyme neuraminidase - structure indicates small (3 A) movement of variable and light chains upon binding
    2. The complex of Fab with HIV-1 peptide shows the largest change
    - This variability means that attempts at using models to predict the conformation of CDR loops is rather less soundly based
  32. 32How do you make monoclonal antibodies?- Produced by B cells - displayed on surface when stimulated by antigen - causes cell to divide- would normally produce polyclonal antibodies
    - But prefer monoclonal antibodies - genetically identical - made from a single clone of a B cell and then immortalise by turning it into a tumour cell
  33. 33How do you humanise mouse antibodies?- Starting with the attempt to humanise mouse anti-lymphocyte CAMPATH-1
    - Put the mouse variable domain onto a human IgG molecule - but remained very immunogenic
    - But if you only use mouse CDRs - grafted onto a IgG framework of the human antibody it is tolerated
  34. 34How can phage display be used to study protein interactions?- Extra DNA inserted into genes that code for coat proteins - appear on outside of the cell
    - Two genes have been found to do this effectively - P 3 which is the minor coat protein and P8 - the major coat protein - has 2700 copies which constitute the bulk of the coat - addition of DNA adds a peptide to the N-terminus of both the proteins
    - If you insert random DNA you can create a massive library - used for panning - take ligand of interest and immobilise on column then you add your library of phage - wash off the proteins that bound, amplify and resequenced - characterise
  35. 35Why is phage display important for antibody cloning?- Normally, antibodies are made from monoclonal B cells and then fused with tumour cells to form hybridoma
    - Can be hard to know which antibody producing hybridoma actually bind to the protein - had to do a screening
    - Phage display is selective - don't have to rely on individual characteristics of all the clones in the library - much faster and selective
    - The whole antibody is too large to display on outside of phage - instead do variable region of a single chain or fab
    - Antibody gene can be derived from B-lymphocytes or can be mad synthetically in vitro
    - Particularly useful as antibodies for different epitopes can be joined together - makes binding more effective - multivalency better than monovalent constructs
    - Has been used to produce HUMIRA - an antibody that targets the tumour necrosis factor a - a proinflammatory cytokine secreted predominantly by monocytes/macrophages - has effects of coagulation, insulin resistance and endothelial function
  36. 36What alternative scaffolds for protein-binding are there and why are they useful?- Many different types of folds are being investigated - 30 folds so far e.g. B bundle, B barrel, 3 a helix
    - Provide different binding surfaces - the protein with the highest kd can be directed by directed evolution e.g. phage display - but they may have different physical and chemical properties to the original antibodies e.g. disulphide bonds, stability in serum, interaction surfaces - useful properties that may make it easier and cheaper to manufacture the protein

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Biology (Lecture notes ) Flashcards on Enzymes , created by Jenna Robinson on 27/05/2017.
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