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Biochemistry > Enzyme Catalysis > Flashcards

Enzyme Catalysis Flashcards

1
Q

What molecule is common reductant and source of hydride ions (H-) in chemistry?
Why is it not common in biology and what is often used instead?

A

Borohydride (BH4-)

BH4- will reduce almost everything, which would cause massive damage in an organism
- NAD(P)H

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2
Q

What is the structure of Nicotinamide Adenine Dinucleotide (NAD(P)H)?

A

2 nucleotides joined by their phosphates
- Top nucleotide bound to nicotinamide ring
- Bottom nucleotide bound to adenine

Can have additional phosphate at bottom - NADPH

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3
Q

Reduced vs oxidised form of NAD(P)H?

A

Reduced has extra H bound to nicotinamide ring - Hydride ion

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4
Q

What are the specific requirements for a hydride transfer? (2 requirements)

A

Distance of ≈3-3.5Å; Van der Waal distance
Angle of 107° between the hydride and where it is attacking

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5
Q

How are the 2 types of Fatty Acid Synthetases (FASs) different in their catalytic architecture?

A

Type 1 - Multiple catalytic domains on 1 or 2 polypeptides; Substrate passed between domains
Type II - Many discrete polypeptides floating around independently of each other; Each one catalyses a different step

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6
Q

How does the Fatty Acid Elongation Cycle start?
- What is formed and lost?

A

Acyl-ACP (Acyl Carrier Protein) is condensed with Malonyl-ACP by β-ketoacyl synthetase
- Forms 3-ketoacyl-ACP
- Lose an ACP and CO2

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7
Q

What happens to 3-ketoacyl-ACP in step 2 of the FA Elongation Cycle?
- What is formed and converted?

A

3-ketoacyl-ACP reduced by β-ketoacyl reductase (BKR)
- Forms 3-hydroxyacyl-ACP
- NAD(P)H converted to NAD(P)+

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8
Q

What happens to 3-hydroxyacyl-ACP in step 3 of the FA Elongation Cycle?
- What is formed and lost?

A

3-hydroxyacyl-ACP dehydrated to by β-hydroxyacyl dehydratase
- Forms Trans-2,3-Dehydroacyl-ACP
- Lose a H2O

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9
Q

What happens to trans-2,3-dehydroacyl-ACP in step 4 of the FA Elongation Cycle?
- What is formed and converted?
- What happens in the end?

A

Trans-2,3-Dehydroacyl-ACP is reduced by Enoyl reductase
- Forms Longer chain acyl-ACP
- NAD(P)H converted to NAD(P)+
- Cycle restarts and builds upon longer chain acyl-ACP

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10
Q

What is ACP?
How are acyl group attached?
How does FA elongation occur on ACP?

A

Acyl Carrier Protein

Acyl group linked via phosphopantetheine arm that is covalently linked to a serine residue on the carrier protein

Successive addition of 2-C units elongates acyl chain

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11
Q

What is the structure of β-Keto Reductase (BKR)? (4 key things)

A

Tetrameric molecule
Central β-sheet
Set of α-helices on either side of the β-sheet
Loop regions at the top of the β-sheet
- Used for substrate binding and recognition of other copies of the polypeptide chain

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12
Q

What residues are there in the active site of BKR? (2 residues)

A

Conserved tyrosine near the nicotinamide ring of the NAD cofactor
Conserved lysine also nearby
- -ve charge of phosphates on NAD interact with positive end of the α-helices dipole

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13
Q

Look at BKR Mechanism; Also describe in words
- Roles of the active site residues

A

A C=O is reduced when a hydroxyl is generated from a keto group with the aid of NAD(P)H and tyrosine

Lysine is not catalytic but instead stabilises intermediate

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14
Q

What is the purpose of Enoyl Reductases in the FA Elongation Cycle?

A

Remove C=C bond

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15
Q

Comment on the sequence identity and structural similarity between BKR and ENR

A

High structural similarity and low sequence identity

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16
Q

Look at ENR Mechanism; Describe in words too

A

Hydride transfer from NADH to C3 at the double bond (C=C)
Rearrangement to form enolate anion intermediate
Proton donation from tyrosine
Enol <-> Keto tautomerisation to give reduced product

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17
Q

Difference between ENR and BKR Mechanism?

A

Catalytic Tyrosine in different location as site of proton donation is different in each reaction scheme

18
Q

How is ENR different between bacteria and humans?

A

Both contain a flexible molecule:

However in bacteria there is more room for this molecule to take up multiple conformations

In animals there is less room for the molecule and it tends to be locked into one of multiple potential conformations

19
Q

Why is ENR a good antibiotic target?

A

Difference between bacterial and animal ENR can be taken advantage of to target only bacterial ENR

20
Q

Diazaborine is an effective ENR inhibitor. What are the downsides? (2 downsides)

A

Bacteria developed resistance rapidly
Boron is essential to the compound but is toxic

21
Q

How does Diazaborine interact with ENR? (hint - sits)
How is resistance developed?

A

Diazaborine sits directly on Nicotinamide ring, blocking hydride transfer
Boron forms covalent link to NAD; Unusual

Mutation turns Glycine into Alanine; Blocks drug

22
Q

Why is triclosan a better antibiotic for bacterial ENR inhibition than Diazaborine? (2 advantages; Give detail)

A

Sits on Nicotinamide ring just like Diazaborine

However, it contains no Boron; No covalent link formed and non-toxic
- Binding effectiveness comes from interactions with environment

Developing resistance to triclosan is much harder
- Bulkier mutations required; These mutations can also block substrate - Trade-off

23
Q

What about triclosan makes it a good mimic of mimic of ENR intermediate (enolate anion)?
What structural elements cause this? (3 elements)

A

Bind with high affinity to the enzyme; EC50 in nM levels

Has a O-/OH group
2Cl ring of triclosan mimics Pantetheine arm of enolate anion
Bottom of triclosan mimics acyl chain

24
Q

The 3rd step of FA Elongation (FAS) involves dehydration. What are the 2 enzymes which can catalyse this?

A

FabZ or FabA

25
Q

What additional reaction can FabA catalyse?

A

Isomerisation reaction to change between E and Z isomers

26
Q

What is the structure of FabZ and FabA? (hint - “hotdog” fold)
What are the conserved residues? (2 residues)
- Distance between them?
- What are the different residues in FabA and FabZ

A

β-sheet wrapping around a central α-helix

Conserved Histidine and Glutamate/Aspartate
- Asp/Glu is distanced from His but both are catalytically involved in mechanism
- FabZ has Glu; FabA has Asp

27
Q

How does FabZ/FabA enzyme function?
What are the 2 pockets for?

A

2 of the enzyme come together to form a dimer; creates 2 active sites with His close to Asp/Glu

Pockets for pantetheine arm and growing alkyl chain

28
Q

Look at FabA/FabZ dehydration mechanism; Describe in words too

A

Removal of proton at C2 by His and donation of proton from Glu/Asp
- Enables hydroxyl at C3 to leave as H2O

Intermediate C1=C2 formed when oxygen of keto group withdraws electrons to itself
- Oxygen then releases electrons and C2=C3 is formed

29
Q

What do nucleases cleave?
At what carbons does this occur?
What type of reaction is this? (hint - abbreviation)

A

Phosphodiester bonds

Either 5’ or 3’; Depends on the enzyme

SN2
- Substitution
- Nucleophilic
- 2 molecules

30
Q

What are the 2 models for the nucleophilic attack of phosphate?
How are they different?

A

Associative - OH nucleophile attacks and then OR leaves

Dissociative - OR dissociates and then OH nucleophile attacks

31
Q

In what conditions does RNA self cleave and why?

A

Alkaline conditions as RNA has an extra OH compared to DNA, and a base can attack this

32
Q

Look at RNA self-cleavage mechanism; Describe in words too (3 steps)

A

In alkaline conditions, a base attacks 2’ OH, removing H
- The O- then attacks the P in phosphate

A base then donates a proton to the 5’ O of the phosphodiester bond
- Breaks the bond

Water donates a hydrogen to either the 2’ or 3’ oxygen; Random
- Restores OH nucleophile

33
Q

How do DNA restriction endonucleases work?
What are the 2 models for how it does this?
What feature enables catalysis?

A

They look for a specific sequence in DNA to latch onto
- They then distort substrate into active site for perfect binding and catalysis

2 models to find sequence:
- Hops along DNA between regions
- Diffuses along DNA; Like train track

Metal ion enables catalysis

34
Q

Similarities in structure of the Type II REases; EcoRV and EcoRI

A

2 identical subunits making a dimer
Central β-sheet flanked by α-helices
‘Cup’ shape so it can land on and scan DNA

35
Q

Role of metal ions in nuclease catalysis? (3 roles)

A

Enhance deprotonation of the attacking water
Stabilise -ve charges on pentavalent phosphate intermediate
Stabilises leaving 3’ oxyanion

36
Q

How does the metal ion in the nuclease MvaI act as a cofactor? (2 ways) (hint - positioning)

A

Increase the ability of the enzyme to do its job
- Positions water molecule to allow it to become nucleophile
- Positions the nucleophile and phosphate substrate so nucleophilic attack can occur

37
Q

Why is Mg2+ used over Mn2+?
What other divalent metal ions could be used?
- Cons
What is one thing about divalent metal ions that is consistent?

A

Magnesium is more abundant so it is favoured

Ca2+ is rare and often has an inhibitory effect

There is 1 consistent position near scissile phosphate always filled by metal ion

38
Q

What is the Role of Mg1 (hint - nucleophile) and Mg2 (hint - water) in 2 metal ion nuclease mechanism?

A

Mg-1:
- Facilitates deprotonation of water by a nearby base
- Help position nucleophilic ion that is generated

Mg-2:
- Interacts with leaving oxyanion
- Positions water molecule for proton donation

39
Q

What are the key residues of HNH nucleases and what are their roles? (2 residues)
What other feature is there in the active site?

A

Histidine - Acts as the attacking base to establish nucleophile
Asparagines - Positions substrate

Mg2+ stabilises transition state

40
Q

What can change between different HNH nucleases?
What is conserved?

A

Metal ion that is used; Can be Ni2+, Zn2+ etc.
- Histidine and cysteine residues bind to Zn2+ for use in a structural (not catalytic) role

Histidine is conserved

Biochemistry (7 decks)

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