(6-8) Co-factors in enzyme mechaisms, Rafferty

What is used as a reductant (source of hydride ions) in Chemistry vs Biology?

• BH4-

• NADP(P)H

When NAD(P)H is in its oxidised form, what is the nicotinamide ring like?

The positive charge is distibuted over the ring

How is hydride transfer on the nicotinamide ring stereospecific?

The enzyme transfering the hydride differentiates between the two hydrogens, it tranfers to one particular face of the nicotinamide ring (top or bottom)

What is crucial about hydride transfer?

Distance and angle of approach (distance 3-3.5A)(C=O at 107’)

What is the difference between Type 1 and type 2 FAS

Type 1 - large multifunctional protein, all catylitic activities on one polypeptide chain (Animals & Fungi)

Type 2 - Sperate indavidual enzymes (Plants & Bacteria)

What is the Acyl carrier protein (ACP)

A transport protein that carries the growing acyl chain

What is lost in the condensation step of the fatty acid elongation cycle?

• ACP

• CO2

What does the enzyme BKR do?

Catalyses the first reductive step of fatty acid elongation cycle

• Requires NADPH (for the H)

• Tetrameric protein

(Beta-keto ACP reductase)

What 2 residues are conserved in BKR?

A tyrosine and a Lysine near the NADPH co factor

What is the BKR reaction mechanism?

A carbon oxygen double bond is reduced when a hydroxyl group is generated from a keto group with the aid of a NAD(P)H cofactor and a conserved tyrosine

What does Enoyl ACP Reductase (ENR) do?

Catalyzes the final reductive step of fatty acid elongation, (removes the double bond in the chain, very hard step for chemists but not biology)

• Requires NAD(P)H

What does Beta-Hydroxyacyl dehydratase do?

Removes a water in the fatty acid elongation cycle, creating a double bond in the chain

How many carbons are added each cylce of the fatty acid elongation cycle?

2

What are steps in the fatty acid elongation cycle?

• 2 carbons added (ACP + CO2 removed)

• Keto reduced (=O → -OH) (NAD(P)H → NAPD)

• Water removed by dehydratase (creates double bond in main chain)

• Double bond removed in main chain by Enoyl reductase (NAD(P)H → NADP)

What are the structual differences/similarities between BKR and ENR?

• Very similar stucturaly, despite low sequence similarity

• Both contain a Rossman fold for binding NADPH

What is the ENR catalytic mechanism?

• A hydride is transfered from NADH to C3 (double bond)

• Rearangement to form enolate anion intermedate

• Tyrosine donates proton

• Enol ←→ keto tautomerzation to give reduced product

What is the difference in the position of the tyrosine residues in ENR and BKR?

• ENR, closer to C1

• BKR, closer to C3 keto

crucial for proton donation

How do Thienodiazaborines inhibit the ENR?

• Boron atom enables the mimicry of the trasition state#

• Forms a coordinate bond with the 2’-OH group of NAD+, trapping it and disabling the enzyme

What sources of resistance might bacteria develop to overcome Thienodiazaborine?

• Mutations in ENR gene (point mutation that prevents Thienodiazaborine from binding/fitting)

• Overexpression of ENR

• Efflux pumps (pump Thienodiazaborine out)

• Reduced permeability to Thienodiazaborine

• Alternative pathways

How does Tricolsan inhibit the ENR (specifical Fabl of E.coli)?

• OH binds tightly to tyrosine and NAD+

• Mimics a substrate intermediate

What is the G93V mutation in Fabl (E.coli)?

• Glysine switched for a Valine

• Valine more bulky and hydrophobic

• changes shape of binding pocket so Triclosan dosent fit but normal enzyme activity can continue

• Good example of bacterial resistance

What are the similarites and differences between Triclosan and Thienodiazaborine?

• Thienodiazaborine has a boron atom, wherase Triclosan has a OH group (that binds)

• Both mimic substrate intermediate

• Triclosan widely used but not favoured anymore due to resistances

• Thienodiazaborine presents new oportunities

What do the FabZ and FabA enzyme do?

Catalyse the dehydration step in FAB

What can FabA also do that FabZ can’t?

Catalyse an aditional isomerisation reaction

What are the structures of FabA and FabZ?

• Hotdog fold, alpha-helix wrapped in a beta-sheet

• Both function as dimers of identical sub-units

• Both have a His residue in active site

• Fab A has a Asp

• Fab Z has a Glu

What is the mechanism of FabA and FabZ?

• Removal of a proton at C2 by His

• Donation of proton by Glu(FabZ) or Asp(FabA) to enable hydroxyl group to leave

• There is an intermediate double bond formed betweenC1 and C2 when the oxygen of the keto group at C1 withdraws the electrons to itself and subsequntly releases them after a C1-C2 double bond is formed

Phosphodiseter bond hydrolysis is an SN2 reaction, what does this mean?

• S = substitution (AB + C = AC +C)

• N = nucleophile, Donate an electron pair, often negatively charged or strongly negative

• 2 = Bimolecular

What happens ,as a result of SN2 attack, to the configuration of the phospahte?

Inversion (bent)

What is the Associative mechanism of phosphodiester hydrolysis?

• When a pentavalent intermedate is formed

• Strong nucleophile, weak leaving group

What is the Dissociative Mechanism of phosphodiester hydrolysis?

• When a trigonal intermediate is formed (PO3-)

• Strong leaving group, weak nucleophile

What are RNA phospodiester bonds suceptible to under alkaline conditions?

Self cleavage

What is the general mode of action of restriction endonuclases?

• Non specific binding

• Linear diffusion (moves along nucleic acid)

• When finds a specific binding side → catlysis occurs

• Product release

What are the 3 things (general) required for phosphodiester bond hydrolysis?

• General base - for deprotonating of the water to make an attacking nucleophile

• Lewis acid (electron pair acceptor) - to stabilise the pentavalent intermediate

• General acid - to protonate the leaving oxyanion

What is the PD…D/ExK motif found in REases like EcoRI and EcoRV?

• P - proline

• D - Aspartate

• E - Glutamine

• x - any residue

• K - lysine

coordinates the Mg2+ ions essential for catylsis

Distance between PD and DExK can vary massively between different REases

What has been proposed as the roles that Metal ions play in REases catalytic process?

• Enhance the deprotonation of the attacking water (nucleophile)

• Stabilise the negative charges of the pentavalent intermediate state of the phosphate

• Could provide some stabilisation to the leaving 3’oxyanion

Which metal ions are used by REases?

• Mainly Mg2+ (Mn2+ would work its just not that abundant)

• Magnesium has a well defined coordination shell that can position water molecules, it also has a high charge density

• Ca2+ is rare, often seen to have inhibitory activity

How does one metal ion catalysis work in phospodiester bond cleavage?

‘substrate assisted cleavage’

The Mg2+ ion only stabilises pentavalent intermediate and helps protonate leaving oxyanion. Not deprotonating the water for a nucleophile. This is done by the adjacent phospho group. (Replacent of the O- with an S or CH4 abolishes cleavage)

How does the two metal ion mechaism work (REases)?

• Mg1 may facilitate deprotonation of a water by a nearby base, and help position it

• Mg1 and Mg2 are idealy 4A apart and both stabilise the negative charge on the pentavalent phosphorus

• Mg2 interacts with the leaving oxyanion and may also position a water molecule for proton donation

What type of nuclease is found in Cas9?

NHN, has a number of conserved His and Asp residues

• uses a histidine as the base to establish the attacking nuclophile

• Uses an Mg2+ ion to stabilize the transition state

What other metal ions have HNH-type nucleases been found to use?

Mg2+,Ni2+,Co2+,Zn2+ even Ca2+