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MBB261 > Rafferty > Flashcards

Rafferty Flashcards

1
Q

What is borohydride used for in chemistry?

A
  • routine reductant and source of hydride ions
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2
Q

Why is there a problem with using borohydride in biological systems?

A
  • boron can be harmful

- borohydride highly nonspecific

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3
Q

What are used as alts to borohydride in biology, and why are they better?

A
  • cofactors, such as NAD(P)H

- much more gentle and precise

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4
Q

What part of NAD(P) is where the important chem happens?

A
  • nicotinamide ring
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5
Q

What position does hydride transfer occur on in NAD(P)?

A
  • C4 position on nicotinamide ring
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6
Q

What is the charge on NAD(P) and where is it located?

A
  • +ve charge distributed over ring

- bit net overall charge is -ve, due to 2 phosphates

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7
Q

What diff sources can NAD(P) come from?

A
  • nicotinic acid digested
  • nicotinamide from breakdown of Trp
  • nicotinamide mononucleotide –> ring system stuck onto ribose, in most systems take and use NAD pyrophosphorylase, which uses ATP to add on adenine-ribose-PO4- and add on 2 PO4- to make complete NAD molecule
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8
Q

What is the diff in shapes of NAD and NADH?

A
  • NAD planar, and NADP not
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9
Q

How do NAD and NADH act as oxidoreductases?

A
  • DIAG*
  • hydride from NADH transferred to C of planar target molecule and one of bonds to O broken
  • O tries to pull off proton from another functional group on surface of enzyme
  • reaction also works in reverse using NAD
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10
Q

How is hydride ion stereospecific?

A
  • enzyme differentiates between 2 hydrogens (pro-R or pro-S) on C where hydride transfer occurring
  • transfers to 1 particular face on nicotinamide ring –> dep on whether sitting above or below substrate
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11
Q

How is distance of approach of hydride ion critical to its transfer?

A
  • NAD(H) cofactor and molecule to which hydride transferred, or from which received typically at approx VDW contact distance (≈ 3-3.5Å)
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12
Q

How is angle of approach of hydride ion critical to its transfer?

A
  • nucleophile attacks C=O at 107° angle, as orbitals lean away from node DIAG
  • vertical is Burgi-Dunitz angle
  • horizontal is Flippin-Lodge angle
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13
Q

What are FA synthetases (FAS) and what is their role?

A
  • complex enzyme system

- carry out de novo biosynthesis

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14
Q

What are the 2 types of FAS and where are they found?

A
  • type I have catalytic domains on 1 or 2 really big polypeptides –> in vertebrates, yeast and some bacteria
  • type II have discrete polypeptides catalysing each enzymatic step –> in plants and many bacteria, inc E. coli and M. tuberculosis
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15
Q

What does chain elongation involve, and in what systems does it occur?

A
  • successive addition of 2C units to S-acyl primed acyl carrier protein (ACP)
  • occurs in type I and II systems
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16
Q

What occurs during the FA elongation cycle?

A
  • DIAG*
  • R group gets longer and longer
  • C=C bond red by enoyl reductase and NAD(P) –> NAD(P)+
  • release of FA products
  • another molecule joined by β-ketoacyl synthetase, prod C=O
  • ketone group on C3 red to hydroxyl by β-ketoacyl reductase and NAD(P)H –> NAD(P)+
  • removal of water by β-hydroxyacyl deHydratase
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17
Q

What is the role of E. coli ACP, and how is the acyl group attached?

A
  • transport protein that carries growing acyl chain

- attached via phosphopantetheine arm covalently linked to Ser32

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18
Q

What does the crystal structure of E. coli acylated ACP show?

A
  • 4 helix bundle
  • lipophilic cavity –> where put growing acyl chain, but can’t stay there all the time, as needs to do chem and undergo elongation
  • ligand/acyl group exists in “bound”/buried and “unbound”/non-buried forms
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19
Q

How is the growing FA chain bound by ACP?

A
  • binding pocket expands to accom growth
  • binding stabilises ACP
  • FA chain and phosphopantetheine arm adopt no. of diff binding modes
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20
Q

What is the role of beta keto reductase (BKR)

A
  • catalyses 1st reductive step of FA elongation cycle
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21
Q

What is BKR dep on?

A
  • NADPH
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22
Q

What is the structure of BKR?

A
  • tetrameric –> 4 active sited indep
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23
Q

What family is BKR a member of?

A
  • short chain deHase-reductase (SDR) family of NAD/NADP dep oxidoreductases
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24
Q

What common critical feature do SDR family of enzymes have?

A
  • Rossman fold and S…YxxxK motif
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25
Q

What is in the active site of BKR, and how is there position important?

A
  • conserved Tyr near nicotinamide ring of NAD cofactor
  • conserved Lys also nearby
  • sit in such way that nicotinamide ring in just right place on surface of enzyme
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26
Q

What is the reaction mechanism of BKR?

A
  • C=O red when hydroxyl group gen from keto group, w/ aid of NAD(P)H cofactor and conserved Tyr
  • DIAG*
  • O starts to withdraw e-s towards itself (δ-), making C attractive centre to put hydride, so hydride transferred to position 3
  • then gives formal -ve charge to O
  • nearest available proton is on end of hydroxyl group of Tyr, ripped away by O
  • overall get neutral compound, as taken H- an H+
  • Tyr takes proton back from water molecule to remain neutral and system resets
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27
Q

Where was ENR (enoyl ACP reductase) isolated from?

A
  • chloroplast of Brassica Napus
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28
Q

What did looking at structure of Brassic napus ENR show?

A
  • tetramer in vitro
  • looking at monomer showed nucleotide binding fold (Rossman fold)
  • looks a lot like BKR
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29
Q

Does Brassic napus ENR use NADH or NAD(P)H?

A
  • NADH specific
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30
Q

What did a comparison of B. napus BKR and ENR show?

A
  • high degree of structural similarity
  • but low seq similarity (<20% identity)
  • active site critical Tyr residues do not directly superimpose, but phenyl hydroxyl groups v close in position
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31
Q

What is the catalytic mechanism of ENR?

A
  • DIAG*
  • hydride transfer from NADH to C3 position at double bond in acyl substrate
  • rearrangement to form enolate anion intermediate
  • proton donation from Tyr sidechain
  • enol keto tautomerisation to give red products (classic resonance pair)
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32
Q

What is the diff in reaction mechanisms of B. napus ENR and BKR?

A
  • in ENR hydride to position 3 and proton onto O attached to position 1
  • in BKR hydride to position 3 and proton onto O attached to position 3
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33
Q

Why are Lys and Tyr closer together in BKR than ENR?

A
  • diff in site of proton donation in respective reaction schemes
  • point of transfer of hydride and proton separated by more bonds in ENR (3) than BKR (1)
  • so Tyr needs to move slightly further away so can be used in ENR and in slightly diff orientation, using Lys as ref point
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34
Q

Why is it debatable whether BKR and ENR are a case of gene duplication?

A
  • as seq identity so low
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35
Q

What is the significance in the distinct diff in structure of enzyme pathways between humans and bacteria?

A
  • offers opportunity to target bacterial system w/ drug compounds that hopefully won’t affect humans in same way
36
Q

Are E. Coli ENR-NAD complex and B, napus ENR similar?

A
  • v similar and high seq identity

- but missing/flexible loop region

37
Q

What is the structure of diazaborine inhibitors of ENR?

A
  • DIAG*
  • X = thieno, benzo, furo, pyrrolo etc.
  • -> always attached to other ring and all had greater or lesser potency as inhibitor of enzyme
38
Q

Why is ENR-thienodiazaborine complex a good inhibitor?

A
  • thieno ring (contains sulphur) sits where expect substrate to bind on top of nicotinamide ring and forms covalent bond to ribose ring form Boron atom
39
Q

How can you get resistance to ENR-thienodiazaborine compounds, and why?

A
  • mutation Gly93
  • Gly93 packs next to SO2 group on diazaborine
  • causes VDW clash if anything bigger than Gly, so ENR-thienodiazaborines can’t bind
  • but doesn’t seem to overly effect binding of natural substrate, so bacteria can mutate and generate resistance to ENR-thienodiazaborines easily
40
Q

How is flexible loop ordering in E. coli ENR-NAD-diazaborine complexes beneficial?

A
  • makes pocket inaccessible to water so chem can continue
41
Q

In what is triclosan often found?

A
  • antibacterial products
42
Q

Why was there concern about triclosan encouraging formation of drug resistant mutants, and did this happen?

A
  • wouldn’t just be resistant to triclosan but also other compounds which may want to use as antibiotics
  • as encouraging them to prod things which may be triclosan like
  • discovered did get resistance mutant and enzyme targeting striking was ENR
43
Q

Is ENR-NAD-triclosan a good inhibitor, and what does this show?

A
  • yes, sits on top of nicotinamide ring and forms structure more like that formed w/ natural substrate
  • no link between triclosan and ribose, even though triclosan much more potent than any other diazaborines formed
  • so covalent bond isn’t what’s important, it’s the comparison to substrate intermediate/transition states
44
Q

What are the consequences of a G93V mutation in E. coli ENR-NAD-triclosan complexes?

A
  • if bigger than Ala, interferes way enzyme interacts w/ natural substrate so mutations unfavourable (even though protects from antibacterial compounds)
  • eg. can put Val in and has inhibitory effect as VDW distances overlap
45
Q

How does triclosan mimic ENR substrate intermediate?

A
  • DIAG*
  • not perfect mimic as missing part of ring
  • ACP equivalent to rest of triclosan structure
46
Q

What is parasitic ENR found, and where was it found?

A
  • gene encoding type II FAS ENR found in P. falciparum

- in essential subcellular organelle, apicoplast (evo from cyanobacteria)

47
Q

What is conserved in parasitic ENR?

A
  • residues involved in triclosan binding
48
Q

What are triclosan derivatives being dev for?

A
  • inhibit bacterial and parasitic ENRs
49
Q

What does structural comp of E. coli ENR and M. tuberculosis InhA (ENR) show?

A
  • InhA appears to have wider substrate binding cleft than E.coli ENR
  • both show covalent mod of NAD cofactors by inhibitors
50
Q

How were a range of diff compounds found to inhibit ENR, and which was selected for further dev?

A
  • start w/ relatively simple compound and elaborate on it
  • or do random screening
  • library of compounds screened virtually based in mol shape templates and electrostatic charge characteristics of 4 template molecules
  • benzimidazole selected after 1st round of screening and assays using Francisella ENR –> w/ bound benzimidazole compound derivative compound enable further structure guided drug dev
51
Q

Why are phosphodiester bonds an asymmetric system?

A
  • 2 ester bonds formed to diff positions on ribose
  • one to 3’ and one to 5’
  • can cut on either side of phosphate and products will be diff
52
Q

What can other enzymes do once phosphodiester bond cleaved?

A
  • cleave off remaining phosphate and leave DNA blunt ended
53
Q

What is cleavage of phosphodiester bond critical to?

A
  • processing of DNA and RNA
54
Q

What is the general scheme for hydrolysis of NAs?

A
  • follows SN2-type nucleophilic attack, which results in inversion of configuration at phosphate (leaving O always opp side to attacking O)
  • DIAG*
  • OH attacks P
  • energy from P-O bond breaking used to pick up proton (usually from water)
  • 3 Os attached in same plane and space either side where pot 2 others can attach
  • hydroxide ion regen
55
Q

What is the diff between associative and dissociative mechanism for NA hydrolysis?

A
  • DIAG*
  • in dissociative approach of hydroxide disturbs OR1 balance w/o binding, leaving group lost before assoc w/ hydroxide group
56
Q

When is RNA phosphodiester bond susceptible to self cleavage?

A
  • under alkaline conditions
57
Q

How does self cleavage of RNA phosphodiester bond occur?

A
  • DIAGS*
  • RNA unstable due to 2’ OH, so if base around can pull off proton and gen O-
  • O- free to attack P
  • then need another group to provide H+ (eg. BH+, H2O, H3O+)
  • so able to pull off proton and add to O to gen leaving group w/ OH on it
  • 1 product has 3’ OH and 2’ phosphate
  • other product has 3’ phosphate and 2’ OH
58
Q

What enzymes cleave RNA?

A
  • nucleases

- RNA may act as enzyme and cleave itself

59
Q

How is DNA cleaved, and why?

A
  • generally req nuclease action

- can’t self cleave

60
Q

Are nucleases separate enzymes?

A
  • can be
  • or can be domains of other bigger enzymes
  • eg. DNA pol I has nuclease domain w/ checking function to chop off bits of added base
61
Q

What is the structure of REases?

A
  • often dimers (hetero/homo)

- DNA binding interface (generally +vely charged)

62
Q

What is the general mode of action of REases?

A
  • bind non specifically, then linear diffusion until find specific seq it cuts
  • then binds specifically
  • coupling occurs –> often changes shape and often bends DNA a bit
  • then catalysis and products released
63
Q

What are the 2 most well characterised type II REases?

A
  • EcoRI and EcoRV
64
Q

What signature motif do EcoRI and EcoRV active sites contain, and how does it vary in diff REases?

A
  • PD…D/ExK motif
  • spacing between PD and D/ExK can vary from 4->50 residues in diff REases –> so much variance that often hard to spot, so structural info often req for full identification
65
Q

How are EcoRI and EcoRV similar/diff?

A
  • not identical in terms of fold, but are in mechanistic approach
  • intermediate part extended out into in diff shape, but both ended up w/ roughly same relationship w/ phosphate?
66
Q

What is a general reaction scheme for restriction endonucleases?

A
  • DIAG*
  • X = general base for deprotonation of water to make attacking nucleophile
  • Y = Lewis acid (e- pair acceptor) to stabilise transition state
  • Z = general acid to protonate leaving oxyanion
67
Q

What is the role of metal ions in REases?

A
  • stabilise -ve charges on pentavalent transition state of phosphate
  • might enhance deprotonation of attacking water (nucleophile)
  • could provide some stabilisation to leaving 3’ oxyanion (don’t provide proton but could help stabilise by being in vicinity of -ve charges)
68
Q

How are metal ions bound in catalytic site of nuclease MvaI?

A
  • water nucleophile bound to 1 of metal ions –> attacks P

- Asp and Glu involved in binding to setting up of 2 metal ions

69
Q

Which metal ions are used, and why?

A
  • preference for Mg2+ as abundant
  • also poss to use Mn2+ but less abundant so less signif
  • Mg has well defined coordination shell that can position water molecules and possesses high charge density
  • but data for other divalent metal ions showing some activity w/ certain nucleases
  • use of Ca2+ seems to be quite rare and often seen to have inhibitory rather than promoting activity –> about right size to fit into pocket but then doesn’t work
70
Q

How many metal ions are used?

A
  • varies between 1 and 3

- seems to be 1 consistent position located near scissile phosphate that is filled by divalent metal ion

71
Q

What is the 1 metal ion mechanism, and how do we know the importance of the adj phosphate?

A
  • “substrate-assisted” cleavage
  • metal ion interacts w/ phosphate and helps stabilise intermediate form
  • phosphate pulls off proton from water and gens OH –> protonates self briefly to allow OH to attack phosphate
  • replacing adj phosphate w/ phosphorothionate or methylphosphonate, decreases or abolishes cleavage by REase
72
Q

What are the roles of each ion in the 2 metal ion mechanism, and how are they related to each other?

A
  • 1st may facilitate deprotonation by nearby base –> or chain of water molecules linked to bulk solvent
  • 2nd interacts w/ leaving oxyanion and may position water molecule for proton donation
  • 2 ions ideally 4Å apart and stabilise double -ve charge on pentavalent transition state
73
Q

Where did the original 2 metal ion mechanism come from?

A
  • studies of 3’ exonuclease domain of DNA pol I
74
Q

What are the roles of each ion in the 3 metal ion mechanism?

A
  • 1st organises nucleophile
  • 3rd stabilises transition state
  • 2nd has indirect structural role and may result from movement of 3rd ion to diff position (ie. same ion, only 2 in total)
75
Q

What are another class of nuclease which use metal ions to facilitate phosphodiester bond cleavage, and what conserved residues do they have?

A
  • HNH-family nucleases

- no. of conserved His and Asn residues

76
Q

What CRISPR system component has a HNH domain and what does it do?

A
  • Cas9
  • uses His as base to establish attacking nucleophile
  • uses Mg2+ to stabilise transition state
77
Q

Can Zn be used as a metal ion, why?

A
  • could be, but issues as coordination completely diff
78
Q

What flap structures are nucleases req to process?

A
  • those found near rep forks when Okazaki fragments are being removed from copied lagging strand
79
Q

Why are flap structures gen in DNA?

A
  • need to remove RNA primers

- happens by driving pol along and bumping into RNA primer and gen flap in DNA

80
Q

How do nucleases process flap structures?

A
  • RNAse H does a lot of trimming and chews down from end until virtually reaches flap point
  • flap endonuclease (FEN) either trims off last bit left behind by RNAse H or comes in at base where flap joins duplex DNA and chops it all off
81
Q

In what organisms are FENs found?

A
  • all kingdoms of life
82
Q

What do mutations and KOs of FEN genes in many lead to?

A
  • genome instability and cell cycle arrest
83
Q

How are FENs encoded?

A
  • bacterial usually encoded as part of DNA pol I
  • euk/archaeal are separate proteins
  • some bacteria (eg. E. coli) have separate dedicated FEN as well as DNA pol I encoded copy
84
Q

What are the structural feature of FENs?

A
  • arch which selects for 5’ ssDNA flaps
  • duplex DNA binding domain
  • active site acidic residues –> ordinate metal ions in 2 distinct binding sites, 1 catalytic (CAT1) and 1 structural (CAT2)
  • 3’ flap binding pocket
  • metal binding sites
85
Q

What are the characteristics of the T5 phage FEN?

A
  • has helical arch through which DNA flap fed and 3 metal ions in this active site region
  • threading of DNA flap places phosphate backbone close to Mg ions in active site
  • 2 catalytically active important metal ions position a water molecule so it can be activated to attack phosphate and make pentavalent transition state before products made

MBB261 (6 decks)

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