Rafferty Flashcards
What is borohydride used for in chemistry?
- routine reductant and source of hydride ions
Why is there a problem with using borohydride in biological systems?
- boron can be harmful
- borohydride highly nonspecific
What are used as alts to borohydride in biology, and why are they better?
- cofactors, such as NAD(P)H
- much more gentle and precise
What part of NAD(P) is where the important chem happens?
- nicotinamide ring
What position does hydride transfer occur on in NAD(P)?
- C4 position on nicotinamide ring
What is the charge on NAD(P) and where is it located?
- +ve charge distributed over ring
- bit net overall charge is -ve, due to 2 phosphates
What diff sources can NAD(P) come from?
- 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
What is the diff in shapes of NAD and NADH?
- NAD planar, and NADP not
How do NAD and NADH act as oxidoreductases?
- 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
How is hydride ion stereospecific?
- 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
How is distance of approach of hydride ion critical to its transfer?
- NAD(H) cofactor and molecule to which hydride transferred, or from which received typically at approx VDW contact distance (≈ 3-3.5Å)
How is angle of approach of hydride ion critical to its transfer?
- nucleophile attacks C=O at 107° angle, as orbitals lean away from node DIAG
- vertical is Burgi-Dunitz angle
- horizontal is Flippin-Lodge angle
What are FA synthetases (FAS) and what is their role?
- complex enzyme system
- carry out de novo biosynthesis
What are the 2 types of FAS and where are they found?
- 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
What does chain elongation involve, and in what systems does it occur?
- successive addition of 2C units to S-acyl primed acyl carrier protein (ACP)
- occurs in type I and II systems
What occurs during the FA elongation cycle?
- 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
What is the role of E. coli ACP, and how is the acyl group attached?
- transport protein that carries growing acyl chain
- attached via phosphopantetheine arm covalently linked to Ser32
What does the crystal structure of E. coli acylated ACP show?
- 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
How is the growing FA chain bound by ACP?
- binding pocket expands to accom growth
- binding stabilises ACP
- FA chain and phosphopantetheine arm adopt no. of diff binding modes
What is the role of beta keto reductase (BKR)
- catalyses 1st reductive step of FA elongation cycle
What is BKR dep on?
- NADPH
What is the structure of BKR?
- tetrameric –> 4 active sited indep
What family is BKR a member of?
- short chain deHase-reductase (SDR) family of NAD/NADP dep oxidoreductases
What common critical feature do SDR family of enzymes have?
- Rossman fold and S…YxxxK motif
What is in the active site of BKR, and how is there position important?
- 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
What is the reaction mechanism of BKR?
- 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
Where was ENR (enoyl ACP reductase) isolated from?
- chloroplast of Brassica Napus
What did looking at structure of Brassic napus ENR show?
- tetramer in vitro
- looking at monomer showed nucleotide binding fold (Rossman fold)
- looks a lot like BKR
Does Brassic napus ENR use NADH or NAD(P)H?
- NADH specific
What did a comparison of B. napus BKR and ENR show?
- 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
What is the catalytic mechanism of ENR?
- 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)
What is the diff in reaction mechanisms of B. napus ENR and BKR?
- 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
Why are Lys and Tyr closer together in BKR than ENR?
- 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
Why is it debatable whether BKR and ENR are a case of gene duplication?
- as seq identity so low
What is the significance in the distinct diff in structure of enzyme pathways between humans and bacteria?
- offers opportunity to target bacterial system w/ drug compounds that hopefully won’t affect humans in same way
Are E. Coli ENR-NAD complex and B, napus ENR similar?
- v similar and high seq identity
- but missing/flexible loop region
What is the structure of diazaborine inhibitors of ENR?
- DIAG*
- X = thieno, benzo, furo, pyrrolo etc.
- -> always attached to other ring and all had greater or lesser potency as inhibitor of enzyme
Why is ENR-thienodiazaborine complex a good inhibitor?
- 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
How can you get resistance to ENR-thienodiazaborine compounds, and why?
- 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
How is flexible loop ordering in E. coli ENR-NAD-diazaborine complexes beneficial?
- makes pocket inaccessible to water so chem can continue
In what is triclosan often found?
- antibacterial products
Why was there concern about triclosan encouraging formation of drug resistant mutants, and did this happen?
- 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
Is ENR-NAD-triclosan a good inhibitor, and what does this show?
- 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
What are the consequences of a G93V mutation in E. coli ENR-NAD-triclosan complexes?
- 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
How does triclosan mimic ENR substrate intermediate?
- DIAG*
- not perfect mimic as missing part of ring
- ACP equivalent to rest of triclosan structure
What is parasitic ENR found, and where was it found?
- gene encoding type II FAS ENR found in P. falciparum
- in essential subcellular organelle, apicoplast (evo from cyanobacteria)
What is conserved in parasitic ENR?
- residues involved in triclosan binding
What are triclosan derivatives being dev for?
- inhibit bacterial and parasitic ENRs
What does structural comp of E. coli ENR and M. tuberculosis InhA (ENR) show?
- InhA appears to have wider substrate binding cleft than E.coli ENR
- both show covalent mod of NAD cofactors by inhibitors
How were a range of diff compounds found to inhibit ENR, and which was selected for further dev?
- 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
Why are phosphodiester bonds an asymmetric system?
- 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
What can other enzymes do once phosphodiester bond cleaved?
- cleave off remaining phosphate and leave DNA blunt ended
What is cleavage of phosphodiester bond critical to?
- processing of DNA and RNA
What is the general scheme for hydrolysis of NAs?
- 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
What is the diff between associative and dissociative mechanism for NA hydrolysis?
- DIAG*
- in dissociative approach of hydroxide disturbs OR1 balance w/o binding, leaving group lost before assoc w/ hydroxide group
When is RNA phosphodiester bond susceptible to self cleavage?
- under alkaline conditions
How does self cleavage of RNA phosphodiester bond occur?
- 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
What enzymes cleave RNA?
- nucleases
- RNA may act as enzyme and cleave itself
How is DNA cleaved, and why?
- generally req nuclease action
- can’t self cleave
Are nucleases separate enzymes?
- 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
What is the structure of REases?
- often dimers (hetero/homo)
- DNA binding interface (generally +vely charged)
What is the general mode of action of REases?
- 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
What are the 2 most well characterised type II REases?
- EcoRI and EcoRV
What signature motif do EcoRI and EcoRV active sites contain, and how does it vary in diff REases?
- 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
How are EcoRI and EcoRV similar/diff?
- 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?
What is a general reaction scheme for restriction endonucleases?
- 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
What is the role of metal ions in REases?
- 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)
How are metal ions bound in catalytic site of nuclease MvaI?
- water nucleophile bound to 1 of metal ions –> attacks P
- Asp and Glu involved in binding to setting up of 2 metal ions
Which metal ions are used, and why?
- 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
How many metal ions are used?
- varies between 1 and 3
- seems to be 1 consistent position located near scissile phosphate that is filled by divalent metal ion
What is the 1 metal ion mechanism, and how do we know the importance of the adj phosphate?
- “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
What are the roles of each ion in the 2 metal ion mechanism, and how are they related to each other?
- 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
Where did the original 2 metal ion mechanism come from?
- studies of 3’ exonuclease domain of DNA pol I
What are the roles of each ion in the 3 metal ion mechanism?
- 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)
What are another class of nuclease which use metal ions to facilitate phosphodiester bond cleavage, and what conserved residues do they have?
- HNH-family nucleases
- no. of conserved His and Asn residues
What CRISPR system component has a HNH domain and what does it do?
- Cas9
- uses His as base to establish attacking nucleophile
- uses Mg2+ to stabilise transition state
Can Zn be used as a metal ion, why?
- could be, but issues as coordination completely diff
What flap structures are nucleases req to process?
- those found near rep forks when Okazaki fragments are being removed from copied lagging strand
Why are flap structures gen in DNA?
- need to remove RNA primers
- happens by driving pol along and bumping into RNA primer and gen flap in DNA
How do nucleases process flap structures?
- 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
In what organisms are FENs found?
- all kingdoms of life
What do mutations and KOs of FEN genes in many lead to?
- genome instability and cell cycle arrest
How are FENs encoded?
- 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
What are the structural feature of FENs?
- 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
What are the characteristics of the T5 phage FEN?
- 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
