Enzyme Catalysis Flashcards
(40 cards)
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?
Borohydride (BH4-)
BH4- will reduce almost everything, which would cause massive damage in an organism
- NAD(P)H
What is the structure of Nicotinamide Adenine Dinucleotide (NAD(P)H)?
2 nucleotides joined by their phosphates
- Top nucleotide bound to nicotinamide ring
- Bottom nucleotide bound to adenine
Can have additional phosphate at bottom - NADPH
Reduced vs oxidised form of NAD(P)H?
Reduced has extra H bound to nicotinamide ring - Hydride ion
What are the specific requirements for a hydride transfer? (2 requirements)
Distance of ≈3-3.5Å; Van der Waal distance
Angle of 107° between the hydride and where it is attacking
How are the 2 types of Fatty Acid Synthetases (FASs) different in their catalytic architecture?
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
How does the Fatty Acid Elongation Cycle start?
- What is formed and lost?
Acyl-ACP (Acyl Carrier Protein) is condensed with Malonyl-ACP by β-ketoacyl synthetase
- Forms 3-ketoacyl-ACP
- Lose an ACP and CO2
What happens to 3-ketoacyl-ACP in step 2 of the FA Elongation Cycle?
- What is formed and converted?
3-ketoacyl-ACP reduced by β-ketoacyl reductase (BKR)
- Forms 3-hydroxyacyl-ACP
- NAD(P)H converted to NAD(P)+
What happens to 3-hydroxyacyl-ACP in step 3 of the FA Elongation Cycle?
- What is formed and lost?
3-hydroxyacyl-ACP dehydrated to by β-hydroxyacyl dehydratase
- Forms Trans-2,3-Dehydroacyl-ACP
- Lose a H2O
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?
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
What is ACP?
How are acyl group attached?
How does FA elongation occur on ACP?
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
What is the structure of β-Keto Reductase (BKR)? (4 key things)
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
What residues are there in the active site of BKR? (2 residues)
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
Look at BKR Mechanism; Also describe in words
- Roles of the active site residues
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
What is the purpose of Enoyl Reductases in the FA Elongation Cycle?
Remove C=C bond
Comment on the sequence identity and structural similarity between BKR and ENR
High structural similarity and low sequence identity
Look at ENR Mechanism; Describe in words too
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
Difference between ENR and BKR Mechanism?
Catalytic Tyrosine in different location as site of proton donation is different in each reaction scheme
How is ENR different between bacteria and humans?
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
Why is ENR a good antibiotic target?
Difference between bacterial and animal ENR can be taken advantage of to target only bacterial ENR
Diazaborine is an effective ENR inhibitor. What are the downsides? (2 downsides)
Bacteria developed resistance rapidly
Boron is essential to the compound but is toxic
How does Diazaborine interact with ENR? (hint - sits)
How is resistance developed?
Diazaborine sits directly on Nicotinamide ring, blocking hydride transfer
Boron forms covalent link to NAD; Unusual
Mutation turns Glycine into Alanine; Blocks drug
Why is triclosan a better antibiotic for bacterial ENR inhibition than Diazaborine? (2 advantages; Give detail)
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
What about triclosan makes it a good mimic of mimic of ENR intermediate (enolate anion)?
What structural elements cause this? (3 elements)
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
The 3rd step of FA Elongation (FAS) involves dehydration. What are the 2 enzymes which can catalyse this?
FabZ or FabA
