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MBB266 > Rice (Proteins, RNase & lysozymes) > Flashcards

Rice (Proteins, RNase & lysozymes) Flashcards

1
Q

What type of amino acids are all natural proteins made up of?

A
  • L
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2
Q

Why is cysteine a good nucleophile?

A
  • when H removed from SH, giving -ve charge
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3
Q

Can ionisation states of polar side chains vary?

A
  • some can
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4
Q

What is the direction of a polypeptide chain?

A
  • N-ter to C-ter
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5
Q

What does a Ramachandran plot show?

A
  • which phi (Φ) and psi (Ψ) angles are generously allowed, allowed and disallowed due to steric clashes
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6
Q

How many diff structures could a 100 residue protein have?

A
  • approx 10^48
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7
Q

Are any AAs allowed in disallowed regions of a Ramachandran plot?

A
  • only Gly, so it is often conserved
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8
Q

How is 2° structure formed?

A
  • 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
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9
Q

What is 3° structure?

A
  • assembly of 2° structures w/ intervening loop regions

- arrangement of all atoms in subunit, α-helices, β-sheets, side chains and add cofactors

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10
Q

What are the further structure subdivisions?

A
  • 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
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11
Q

What is 4° structure?

A
  • 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)
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12
Q

How can protein structure be visualised?

A
  • 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
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13
Q

How is X-ray crystallography carried out?

A
  • 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
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14
Q

What is resolution?

A
  • level of detail that can be seen in given map
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15
Q

How high a resolution is need to clearly resolve 2 C atoms?

A
  • better than 1.5Å as this is the length of C-C bond
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16
Q

What detail of a protein can be seen at different levels of resolution?

A
  • 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
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17
Q

How is free energy calculated?

A
  • ΔG = ΔH - TΔS
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18
Q

What are the 4 types of interaction that affect enthalpy from strongest to weakest?

A
  • 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
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19
Q

Is enthalpy or entropy more influential in protein folding?

A
  • ΔHfolding = approx 0 (as unfolded protein chain still makes many interactions)
  • ∴ entropy is key to protein folding
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20
Q

Does entropy take into account entropy of water molecules, and why?

A
  • 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
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21
Q

How do proteins recognise binding partners?

A
  • 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)
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22
Q

Why is shape complementarity important in mol recognition?

A
  • max poss no. interactions
  • clefts in enzymes that complement substrate shape
  • shapes of binding partner complement each other
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23
Q

What do catalysts do?

A
  • decrease energy barrier to forward and reverse reactions in pathway
  • don’t alter eq
  • unchanged after reaction
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24
Q

How do catalysts enhance the reaction rate?

A
  • 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
25
Q

What is general acid-base catalysis?

A
  • 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
26
Q

What is a nucleophile?

A
  • e- donor

- term used if bond formed to something other than H

27
Q

What is ribonuclease A (RNase A) and what is its role?

A
  • 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)
28
Q

What did solution studies of RNase A show?

A
  • 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)
29
Q

What did kinetic studies of RNase A show?

A
  • 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
30
Q

How was chemical modification used to investigate RNase A?

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
31
Q

What is the 3° structure of RNase A?

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
32
Q

What residues are in the specificity pocket of RNase A?

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
33
Q

What does the specificity pocket of RNase A recognise and why?

A
  • 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

34
Q

How was the substrate visualised in the active site of RNase A?

A
  • substrate analogues
  • as dinucleotides or longer RNA molecules cleaved by enzyme
  • used non cleavable compound, UpCH2A
35
Q

What is the proposed reaction mechanism for RNase A?

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
36
Q

What does the RNase reaction create (in order)?

A
  • cyclic 2’-3’ P intermediate
  • 3’P product
  • 5’ OH product
37
Q

What is angiogenin and what is its role?

A
  • medically important homologue of RNase A

- promotes dev of blood vessels in healthy tissues and tumours

38
Q

What is the structure of angiogenin, how was it solved and what is this being used for?

A
  • cells cluster in tubular structure
  • structure solved w/ human inhibitor protein
  • structure being used to design smaller inhibitors as pot anti-tumour agents
39
Q

How many diff classes of RNase is there?

A
  • approx 100

- eg. RNase L (destroys all RNA in cell)

40
Q

What is the role of lysozymes?

A
  • glycosidase enzymes involved in 1st line of defense against bacterial attack
  • cleave peptidoglycan (so little effect on gram -ve bacteria)
41
Q

Where are lysozymes found in humans?

A
  • most bodily secretions, inc tears and nasal mucus
42
Q

What is the difference between gram +ve and gram -ve bacterial cell walls?

A
  • 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
43
Q

What is the structure of peptidoglycan?

A
  • 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
44
Q

How is poly (NAG-NAM) linked and where is it cleaved?

A
  • linked NAM β(1-4) NAG β(1-4)

- lysozyme cleaves NAM β(1-4) NAG linkage

45
Q

What is the diff between NAG and NAM?

A
  • NAM larger, has CH3CHC=O and variable group which can be a hydroxyl or amino of peptide cross link
46
Q

What is the structure of lysozymes?

A
  • 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
47
Q

What is the substrate of lysozymes and where does it bind?

A
  • triNAG

- top half of cleft (=active site)

48
Q

How did the substrates of lysozymes help work out its structure?

A
  • 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
49
Q

How was the structure of the lysozyme active site cleft worked out?

A
  • 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)
50
Q

What are the 2 catalytic groups found in D/E of the lysozyme active site cleft?

A
  • Glu35 and Asp52

- CA functional groups

51
Q

Why are Glu35 and Asp52 important residues in lysozyme active site cleft?

A
  • 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)
52
Q

What was the initial proposed mechanism for lysozyme activity, (by Phillips - now believed to be wrong)?

A
  • 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
53
Q

What is the alt mechanism proposed for lysozymes (nucleophilic sp52 mechanism(?

A
  • 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
54
Q

What are the 2 ways nucleophilic substitution can proceed?

A
  • SN1 = both inversion and retention

- SN2 = inversion only

55
Q

How was only the acyl enzyme intermediate trapped?

A
  • expected enzyme trapped in inactive E35Q mutant of lysozyme in complex w/ substrate analogue, w/ F atom as good leaving group
56
Q

What is the now accepted mechanism for lysozymes?

A
  • 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
57
Q

How do other lysozymes differ?

A
  • 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
58
Q

What are the different types of glycosidases and their roles?

A
  • 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)
59
Q

How similar are the diff types of glycosidases?

A
  • unrelated in seq and structure

- most use v similar catalytic mechanisms involving 2 carboxylate side chains acting as acid and base

MBB266 (7 decks)

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