Karteikarten
Enzymes
Karten in diesem Stapel (36)
- 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< - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 ) Karteikarten am Enzymes , erstellt von Jenna Robinson am 27/05/2017.
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