Rice (Proteins, RNase & lysozymes) Flashcards
What type of amino acids are all natural proteins made up of?
- L
Why is cysteine a good nucleophile?
- when H removed from SH, giving -ve charge
Can ionisation states of polar side chains vary?
- some can
What is the direction of a polypeptide chain?
- N-ter to C-ter
What does a Ramachandran plot show?
- which phi (Φ) and psi (Ψ) angles are generously allowed, allowed and disallowed due to steric clashes
How many diff structures could a 100 residue protein have?
- approx 10^48
Are any AAs allowed in disallowed regions of a Ramachandran plot?
- only Gly, so it is often conserved
How is 2° structure formed?
- if some phi-psi angles occur several times in succession, forms helical structure w/ recurring patterns of H bonds
- H bonds between main chain NH and CO
- stabilise α-helices and β-strands
What is 3° structure?
- assembly of 2° structures w/ intervening loop regions
- arrangement of all atoms in subunit, α-helices, β-sheets, side chains and add cofactors
What are the further structure subdivisions?
- structure motifs = arrangement of few helices and/or strands that occur often in diff structures
- super-2° structures = eg. β-α-β motif in TIM barrell
- domains = distinct sub division of protein
What is 4° structure?
- self assoc into assemblies of several polypeptide chains
- homo-oligomers = where many copies of same polypeptide assembles into dimers trimers etc.
- hetero-oligomers = where copies of diff chains assemble (eg. Hb is α2β2 tetramer)
How can protein structure be visualised?
- 1000x too small to see w/ light microscopy (atoms separated by approx 1Å)
- mainly X-ray diffraction from protein crystals –> prod e- density map
- NMR spec
- high res cryo e- microscopy
How is X-ray crystallography carried out?
- crystals of highly purified target molecule grown and exposed to X-rays to give diffraction pattern
- 3D e- density maps made by measuring diffraction spots
- quicker now –> computer analyses structure
What is resolution?
- level of detail that can be seen in given map
How high a resolution is need to clearly resolve 2 C atoms?
- better than 1.5Å as this is the length of C-C bond
What detail of a protein can be seen at different levels of resolution?
- low = no details of side chains or atomic interactions, but α helices visible as sausages of density
- medium = turns of helix, side chains as blobs but not individual atoms
- high = holes in aromatic rings, almost see individual atoms
How is free energy calculated?
- ΔG = ΔH - TΔS
What are the 4 types of interaction that affect enthalpy from strongest to weakest?
- disulphide bonds (approx -167kJ/mol)
- -> covalent interaction
- -> in extracellular proteins to increase stability in hostile envs
- ionic interactions (approx -15kJ/mol)
- -> electrostatic interactions between opp charges
- -> relatively small no. in any protein
- -> much stronger if buried in centre but charged groups almost always on exterior to H bond w/ water
- H bonds (approx -5 to -15kJ/mol)
- -> electrostatic in origin
- -> v common (100s)
- -> highly directional (dictate protein structure)
- -> interior (in 2° structures) and exterior (to water, ligands and other protein surfaces)
- VdW interactions (approx -1 to -4kJ/mol)
- -> any atom in contact w/ another
- -> dipole-dipole
- -> dipole-induced dipole
- -> London dispersion forces (transitory dipoles)
- -> 1000s in any protein
- -> weak but cumulatively important
Is enthalpy or entropy more influential in protein folding?
- ΔHfolding = approx 0 (as unfolded protein chain still makes many interactions)
- ∴ entropy is key to protein folding
Does entropy take into account entropy of water molecules, and why?
- no
- in unfolded protein, many aromatic and methyl groups exposed to solvent
- water molecules form highly ordered cages around these non polar groups
- in folded proteins aromatic and methyl groups bury themselves in interior and water free to move around in solvent
- ∴ exposed non polar groups entropically disfavoured (=hydrophobic)
- “hydrophobic effect” outweighs loss of entropy
How do proteins recognise binding partners?
- H bonds give specific interactions
- groups of hydrophobic residues on surface give ‘sticky patches’ that can interact w/ hydrophobic ligands or patches on other proteins, releasing ordered waters
- VdW interactions
- ionic interactions often important (eg. basic AAs w/ DNA phosphate groups)
Why is shape complementarity important in mol recognition?
- max poss no. interactions
- clefts in enzymes that complement substrate shape
- shapes of binding partner complement each other
What do catalysts do?
- decrease energy barrier to forward and reverse reactions in pathway
- don’t alter eq
- unchanged after reaction
How do catalysts enhance the reaction rate?
- binding effects = bring substrates close and hold them in optimal orientation to react, stabilise transition states and intermediates
- from substrate to product, all reactions pass through short lived transition states and longer lived intermediates
What is general acid-base catalysis?
- proton transferred while going to/from transition state
- most reactions involve at least 1 proton transfer step (usually many more)
- some side chains can act as acid or base, depending on pH or env
What is a nucleophile?
- e- donor
- term used if bond formed to something other than H
What is ribonuclease A (RNase A) and what is its role?
- digestive enzyme prod in pancreas
- cleaves ss RNA in lower intestine into smaller nucleotide fragments
- RNase 5 is clipped form and also active
- reaction in 18O enriched solution showed P-O5’ bond cleavage
- specific to cut after pyrimidine (U/C)
What did solution studies of RNase A show?
- identified key catalytic intermediate, a 2’-3’ cyclic nucleotide, isolated and characterised
- intermediate divided RNase reaction into 2 steps
1) formation of 2’-3’ cyclic compound
2) cleavage (revealed pH dependence)
What did kinetic studies of RNase A show?
- 2 intersecting curves for pH profile suggest 2 groups titrate
- His titrates around pH6-7, so suggested 2 His in active site –> 1 could act as general acid and 1 as general base
How was chemical modification used to investigate RNase A?
- used reactive compounds to chemically mod key groups in enzyme
- 1 mod attempted was treatment w/ iodoacetate, expected to covalently mod SH groups of Cys, caused loss of enzymatic activity
- wrongly concluded RNase had essential Cys residue (Cys actually caused conformational change in real active site elsewhere in molecule)
- seq of mod RNase showed was unusually reactive His mod
- conclusions were His12 and His119 key residues in active site, close together as only 1 mod not both, 1 could could act as general base and 1 as general catalyst
- showed residues hyperactive only at relevant pHs and in folded conformation
- both residues req to allow mod to occur, so modifying agent must be aligned between 2 key residues
What is the 3° structure of RNase A?
- 3 stranded, V shaped antiparallel β-sheet and 3 short α-helices
- polypeptide chain cross linked by 4 S-S bridges involving residue pairs
- active site is deep cleft, containing essential residues
- RNA substrate binds along bottom of V
What residues are in the specificity pocket of RNase A?
- active site residues inc His12 and His119 directly involved in mechanism, Lys41 stabilises -ve phosphate in intermediate, other basic residues assist RNA binding
- specificity pocket residues inc Phe120 which makes VdW contacts w/ RNA base, Ser123 and Thr45 which H bond
What does the specificity pocket of RNase A recognise and why?
- pocket too small for purines so doesn’t recognise them
- will recognise either pyrimidine –> diff H bonding for U and C, use of Ser or Thr OH groups to H bond
How was the substrate visualised in the active site of RNase A?
- substrate analogues
- as dinucleotides or longer RNA molecules cleaved by enzyme
- used non cleavable compound, UpCH2A
What is the proposed reaction mechanism for RNase A?
- 1st half = conversion of NA to cyclic intermediate, His12 acts as acid, His119 as base
- 2nd half = cleavage of cyclic intermediate, His roles reversed
- at end His states returns to initial config for next cycle
What does the RNase reaction create (in order)?
- cyclic 2’-3’ P intermediate
- 3’P product
- 5’ OH product
What is angiogenin and what is its role?
- medically important homologue of RNase A
- promotes dev of blood vessels in healthy tissues and tumours
What is the structure of angiogenin, how was it solved and what is this being used for?
- cells cluster in tubular structure
- structure solved w/ human inhibitor protein
- structure being used to design smaller inhibitors as pot anti-tumour agents
How many diff classes of RNase is there?
- approx 100
- eg. RNase L (destroys all RNA in cell)
What is the role of lysozymes?
- glycosidase enzymes involved in 1st line of defense against bacterial attack
- cleave peptidoglycan (so little effect on gram -ve bacteria)
Where are lysozymes found in humans?
- most bodily secretions, inc tears and nasal mucus
What is the difference between gram +ve and gram -ve bacterial cell walls?
- gram +ve = inner pm surrounded by thick peptidoglycan (10-20 layers) cell wall
- gram -ve = inner pm surrounded by thin peptidoglycan (1-3 layers) cell wall, covalently linked to lipoproteins in outer membrane
What is the structure of peptidoglycan?
- long polysaccharide chains of alt NAG (N-acetylglucosamine) and NAM (N-acetylmuramic acid) sugars
- cross linked in 3D by polypeptide chains containing unusual AAs (some D AAs)
- entire cell wall 1 enormous bag shaped molecule
- lysozyme cleaves specifically between NAM and NAG in regions where not too many crosslinks
How is poly (NAG-NAM) linked and where is it cleaved?
- linked NAM β(1-4) NAG β(1-4)
- lysozyme cleaves NAM β(1-4) NAG linkage
What is the diff between NAG and NAM?
- NAM larger, has CH3CHC=O and variable group which can be a hydroxyl or amino of peptide cross link
What is the structure of lysozymes?
- polypeptide chains = 129 AAs, 4 S-S bridges
- 2 domains separated by deep cleft
- small β-sheet of mainly hydrophobic residues
- hydrophobic core surrounded by short α helices
What is the substrate of lysozymes and where does it bind?
- triNAG
- top half of cleft (=active site)
How did the substrates of lysozymes help work out its structure?
- substrates are (NAM-NAG) cell walls and poly(NAG) chitin
- no further increase in hydrolysis after adding 6x NAG
- suggests active site will bind 6 sugars
- (NAG)6 and (NAG-NAM) modelled into structure
How was the structure of the lysozyme active site cleft worked out?
- cleft between 2 domains observed to bind triNAG in crystals
- triNAG bound in sites A, B and C
- remaining 3 sugars D, E and F built in by modelling
- complex patterns of interactions –> h bonds to O and N atoms on edges of sugars and hydrophobic/VdW interactions w/ faces of sugars
- NAM can only fit in B, D and F
- hexaNAG cleaved to (NAG)4 and (NAG)2
- NAM-NAG cleavage point must be between D(NAM) and E(NAG)
What are the 2 catalytic groups found in D/E of the lysozyme active site cleft?
- Glu35 and Asp52
- CA functional groups
Why are Glu35 and Asp52 important residues in lysozyme active site cleft?
- at lysozymes optimal pH (approx 6) expect both to be carboxylate ions
- Glu35 still largely protonated (pKa=6.5) as in hydrophobic miroenv, so uncharged
- Asp52 normal and -vely charged (pKa=3.5)
What was the initial proposed mechanism for lysozyme activity, (by Phillips - now believed to be wrong)?
- buried Glu35 donates proton and sugars E-F diffuse away (1st product)
- Asp52 stabilises distorted carbonium ion intermediate in site D
- attack by water –> OH added to Cl of D and H+ to Glu35
- sugars A-B-C-D is 2nd product
- problem is high energy of proposed intermediate
- binding of triNAG lactone supports mehcanism
What is the alt mechanism proposed for lysozymes (nucleophilic sp52 mechanism(?
- involving enzyme-glycosyl covalent bond, rather than carbonium ion intermediate
- v difficult to decide as intermediates similar and only around v briefly
- this mechanism favoured by enymologists
What are the 2 ways nucleophilic substitution can proceed?
- SN1 = both inversion and retention
- SN2 = inversion only
How was only the acyl enzyme intermediate trapped?
- expected enzyme trapped in inactive E35Q mutant of lysozyme in complex w/ substrate analogue, w/ F atom as good leaving group
What is the now accepted mechanism for lysozymes?
- nucleophilic attack by Asp52 forms covalent acyl-enzyme intermediate
- Glu35 donates H+ and E-F diffuse away (1st product)
- attack by water –> OH added to Cl of O and H+ to Glu35
- A-B-C-D is 2nd product
How do other lysozymes differ?
- destruction of bacterial cell walls important function, so other enzymes evolved to carry out same process
- no seq similarity –> but all have mechanisms using 2 carboxyl groups in same relative positions to cleave polysaccharide substrate
What are the different types of glycosidases and their roles?
- lysozymes = break down bacterial cell walls (defense)
- lactases = break down lactose (nutrient acquisition)
- amylases = break down starch
- cellulases = break down cellulose to glucose
- neuraminidases = used by viruses and bacteria to penetrate cell walls (pathogenesis)
How similar are the diff types of glycosidases?
- unrelated in seq and structure
- most use v similar catalytic mechanisms involving 2 carboxylate side chains acting as acid and base
