Fatty acid Breakdown/Synthesis

Question 1

Where does Fatty acid breakdown/b-oxidation occur in the cell?
Where do they come from?

Answer 1

In the mitochondrion
Fatty Acids → Acetyl-CoA

Origins:
1. Uptaken into the cells from circulation in form of lipoproteins
2. Lipolysis (stored in lipid droplets inside the cell
3. Phospholipase A1 & A2 (minor)

Question 2

What is Lang’s cycle?

Answer 2

Phospholipids → {Phospholipase A2} → Lysophospholipd (lose hydrocarbure chain)

*In most cases, FA are reused to go back to phospholipid, so doesn’t really release a FA for breakdown (b-oxidation)

Question 3

How do Fatty acids get into the mitochondria?

Answer 3

Problem: Long chain of FA (>16C) can’t cross the inne mitochondrial membrane

1. Activation by CoA via Acyl-CoA Synthetase
   - high-energy
   - Uses 1 ATP → reaction driven PPi hydrolysis
   - Keeps FA inside the cell + graident to allow more FA in
   - Acyl-CoA is bound to the outer mitochondrial membrane
2. Coupling to Carnitine vis Carnitine Palmitoyl Transferase I (CPT I) → RATE-LIMITING STEP
   - CPT I on outer mito membrane facing cytosol
   - Transfers Acyl from CoA → Acylcarnitine (high-energy) → makes Acylcarnitine
3. Transit towards mitochondrial matrix via Carnitine:acylcarnitine translocase
   - Carnitine out
   - Transported freely across the outer membrane, translocase found in the inner membrane
4. FA release into matrix via CPT II → regenerates Acyl-CoA inside the matrix
   - CPT II located on matrix side of inner membrane
   - Carnitine is recycled to the cytosol
   - Acyl-CoA → b-oxidation → Acetyl-CoA
   - CoA prevents going back in the cytosol

Question 4

What is the rate limiting step of Fatty Acid breakdown?
How is it regulated?

Answer 4

Fatty acid breakdown is limited by substrate availability → entry of Fatty acids in the mitochondrion

Acyl-CoA → {CPT I} → Acylcarnitine → transport into mitochondrion

Inhibited by high [malonyl-CoA] in the cytosol:
- High energy state of the cell
- Stimulates biosynthesis of FA, must stop breaking down
- Binds in the same site of CPT I as Acyl-CoA

Steps:
1. CPT1 → Aceylcarnitine
2. Diffuses through outer membrane
3. IMM transporter Carnitine:acylcarnitine translocase
4. CPT2 inside the matrix converts back to acyl-CoA

Question 5

What are the steps for b-oxidation of saturated FA?

Answer 5

*Start with Acyl-CoA
1. Dehydrogenation → makes FADH2 → ETFreduced → QH2 (Ox. Phos.)
- Oxidizes the C at the b-position (makes double bond between a and b Carbons)
2. Hydration
3. Dehydrogenation → generates NADH
4. Thiolic Cleavage → releases 1x Acetyl-CoA (-2C) and add a new CoA to the end of the new chain (2C shorter)
5. Repeat until no Carbons left

Question 6

What is the balance sheet of b-oxidation for a 16:0 fatty acids like?

Answer 6

16:0 = saturated 16C fatty acid
*Palmotoyl-CoA = Acyl-CoA
1. Activation of palmitate to palmotoyl-CoA → -2 ATPs
2. Oxidation of 8 acetyl-CoA (CAC) → 8x10 = 80 ATP
3. Oxidation of 7 FADH2 → 7x1.5 = 10.5 ATP
4. Oxidation of 7 NADH → 7x2.5 = 17.5 ATP
Net: Palmitate → CO2 + H2O = 106 ATP

Question 7

What are the steps for b-oxidation of unsaturated FA (odd) ?

Answer 7

*Dehydrogenation replaced by isomerisation → no FADH2 produced
1. Isomerization cis → trans double bond
2. Hydration (double bond becomes C-OH)
3. Dehydrogenation
4. Thiolic cleavage
5. Repeat
*For each odd unsaturation → no FADH2 (-1.5 ATP)

Question 8

What is the cost of even vs odd unsaturations?

Answer 8

ODD unsaturations → no FADH2 produced (isomerization instead)
EVEN unsaturations → costs 1 NADPH/even unsaturations
- produce FADH2 (2 double bonds) → reductase (costs NADPH) → ismoerase → good to go

Question 9

What are the general steps of b-oxidation of Odd-chain fatty acids?

Answer 9

1. Normal b-oxidation until last 3 carbons → last 3 carbons released as propinoyl-CoA
   - Last 5 Carbons → Propionyl-CoA + Acetyl-CoA
2. Propionyl-CoA (3C) converted to succinyl-CoA (4C)
3. Succinyl-CoA → CAC

Question 10

Which reactions allow conversion of Propionyl-CoA → Succinyl-CoA

Answer 10

Propionyl-CoA → S-Methylmalonyl-CoA (high-energy intermediate)
- Uses ATP + HCO3- (which is transported on malonyl-CoA)
- Irreversible
- Catalysed by carboxylase
…
→ Succinyl-CoA

Question 11

What are the steps of b-oxidation of branched-chain FA?

Answer 11

Broken down into Propionyl-CoA for branched sections (3C) and Acetyl-CoA for unbranched sections (2C)

Question 12

Which 2 branched chain fatty acids do we generally find in humans?

Answer 12

*Mostly in milk
1. Phytanic acid (20 C)
2. Pristanic acid (19 C)

Question 13

What are the 3 ketone bodies found in humans?

Answer 13

1. Acetoacetate
2. Acetone
3. b-Hydroxybutarate

*Acetyl-CoA can be used to regenerate Ketone bodies
*Wat to deliver water-soluble “fat derivatives” to another tissue

Question 14

When/Where does ketogenesis occur?

Answer 14

*Primarily in the liver
During fasting/starvation, when [glucose] is low → Oxaloacetate is depleted → CAC stops
Problem: Acetyl-CoA doesn’t transfer between tissues
Solution: Ketone bodies serve as “shuttles” for Acetyl-CoA between tissues → bring Acetyl-CoA to the heart and brain so they can run their CAC by regenerating Acetyl-CoA by Thiolase reaction

*Oxaloacetate stores are used for gluconeogenesis as body tries to keep ~5.5mM [glucose]

Question 15

What reactions are involved in ketogenesis?
How do we go from 1 ketone body to another?

Answer 15

Acetyl-CoA → Acetate
Acetyl-CoA + Acetate → Acetoacetyl-CoA → HMG-CoA → {HMG-CoA lyase} → Acetoacetate

Ketone bodies:
Acetoacetate → {dehydrogenase} → b-Hydroxybutarate
Acetoacetate → {release CO2} → Acetone

Question 16

What reactions in the heart and brain allows for conversion of ketone bodies back to Acetyl-CoA?

Answer 16

b-Hydroxybutarate → Acetoacetate → {Succinyl-CoA → Succinate) → Acetoacetyl-CoA → {add CoA-SH} → 2x Acetyl-CoA

*Each ketone body contains 2 Acetyl groups → 2x Acetyl-CoA is have the CoA available

*No acetone can be consumed because goes to the lungs → wasted breath

Question 17

What are outputs of b-oxidation?

Answer 17

1. Acetyl-CoA
2. FADH2
3. NADH

Question 18

Which metabolic circumstances require the synthesis of fatty acids?

Answer 18

• To initiate cell division
• After a glucose-rich meal
• After a fructose-containing meal

Question 19

In which cell compartment does Fatty Acid synthesis occur?

Answer 19

In the cytosol → need to shuttle the Acetyl-CoA from the mitochondria to the cytosol, because it can’t cross with its CoA group

Question 20

How is Acetyl-CoA transported from the mitochondria to the cytosol for Fatty Acid synthesis?

Answer 20

Through the Citrate Shuttle:
1. Acetyl-CoA + Oxaloacetate → {Citrate Synthase} → Citrate Releases the CoA group
2. Citrate/Pi Antiport across the mitochondrial membrane to the cytosol
3. Citrate → {Citrate Lyase, ATP + CoA-SH} → Acetyl-CoA + Oxaloacetate
4. Acetyl-CoA used for FA synthesis + Oxaloacetate → Malate → Pyruvate → transported back to mitochondria

Question 21

What happens to Oxaloacetate when Acetyl-CoA is released in the cytosol after being shuttled as citrate?

Answer 21

*No oxaloacetate transporter
Oxaloacetate (CYTOSOL) → {Malate dehydrogenase, NADH} → Malate → {Malic enzyme, NADP+} → Pyruvate → shuttled back to the mitochondria → {Pyruvate carboxylase, ATP + CO2} → Oxaloacetate (MITO)

NADPH produced goes to Fatty Acid synthesis

Question 22

What is the role of Acetyl-CoA Carboxylase (ACC)?
What is its cofactor?

Answer 22

Converts Acetyl-CoA (2C) → Malonyl-CoA (3C)
- Irreversible, Rate-limiting step of FA synthesis
- Driven by ATP breakdown which makes it exergonic
- 1 enzyme/2 reactions
- Biotin = cofactor which provides Carbon

*Reminder, PEP and PEPCK uses biotin as carbon provider

Question 23

Which reaction is an activation step of fatty acid synthesis?

Answer 23

Adding ACP to Acetyl-CoA (by MAT enzyme)

Question 24

Malonyl/Acetyl-CoA Transacylase?
What is ACP?

Answer 24

Couples acetyl-CoA/Malonyl-CoA with ACP → keeps acetyl and malonyl “activated” → prevents leakage to other compartments
*Switches the CoA for ACP group → primed molecule is now ready for elongation
ACP = Acyl Carrier Protein (similar to CoA)

Question 25

What molecule is made by Fatty Acyl Synthase ?
What are the steps?

Answer 25

Palmitate (C16:0) → used as a basic building block for other fatty acids

1. Acetyl-ACP + Malonyl-ACP →… → Butyryl-ACP
2. Butyryl-KS (4C) + Malonyl-ACP (3C) - CO2 → Acetoacetyl-ACP → … → Butyryl-ACP (goes in cycles 7x)
   → Palmitoyl-ACP → Palmitate

*2 NADPH invested/2C added (/Malonyl-ACP)

Question 26

What is the structure of Fatty Acyl Synthase?

Answer 26

• 1 protein with many domain → all catalytic activities on 1 monomer
• 2 domains make thioester bonds (KS and ACP (main one))
• 2 homodimers organized in upside-down orientation → domains interact with each other
  VERY FAST

Question 27

What is the general stoichiometric equation of the synthesis of Palmitate?

Answer 27

1. 7 malonyl-CoA are derived from Acetyl-CoA:
   7 Acetyl-CoA + 7 CO2 + 7 ATP → 7 malonyl-CoA + 7 ADP + 7 Pi + 7 H+
   *CO2 are released in 1st step
2. Overall Palmitate biosynthesis (by Fatty Acyl Synthase):
   8 Acetyl-CoA + 14 NADPH + 7 ATP → palmitate + 14 NADP+ + 8 COA + 6 H2O + 7 ADP + 7 Pi

Question 28

Which reactions allow elongation of Palmitate to form longer Fatty Acids? (Where does it occur?)

Answer 28

*In the mitochondria → Addition of Acetyl-CoA to palmitate (reverse of FA oxidation)

• Consumes 1x NADH + 1x NADPH
• Adds 2C/ cycle
• Many different enzymes (not like Fatty Acyl Synthase)

Question 29

How is ACC regulated?

Answer 29

*By polymerisation and phosphorylation

Allosteric:
- Activated by Citrate → promotes polymerization
- Inhibited by fatty acids

Hormonal:
Activated by Insulin → stimulates glucose uptake + pyruvate dehydrogenase (more Acetyl-CoA → more Citrate → allosteric polymerization)
→ Dephosphorylation of ACC by Insulin pathway

Glucagon/Epinephrine → inhibits (by PKA-mediated phosphorylation)

Question 30

Which reactions allow desaturation of palmitate or longer fatty acids?

Answer 30

Only 4 different terminal desaturase in mammals → ∆9, ∆6, ∆5 and ∆4 Fatty Acyl-CoA Desaturases
*No desaturase beyond ∆9 in mammals

Question 31

What is the consequence of mammals having no desaturase beyond ∆9?

Answer 31

Some essential fatty acids can’t be produced so need to be acquired from diet (plants)
- Linoleic acid
- Linolenic acid

Question 32

What is the rate limiting step of fatty acid synthesis?

Answer 32

Acetyl-CoA Carboxylase (ACC) → produces Malonyl-CoA

Question 33

What type of reaction in Citrate Lyase from the point of view of the CAC?

Answer 33

Cataplerotic reaction → depletes the CAC
Citrate → Acetyl-CoA
*Produces Acetyl-CoA which is not an intermediate of the CAC
*Cytosolic enzyme

Question 34

What is the importance of fatty acids in immune response?

Answer 34

Some FA are precursor molecules for inflammatory mediators

Pharmacological NSAID (non-steroidal anti-inflammatory drugs) inhibit production these mediators from fatty acids

Question 35

What are some of the general COX inhibitors?

Answer 35

COX = cyclooxygenase: Arachidonic acid (FA) → Prostaglandin F2 (involved in inflammatory response)

COX inhibitors prevent inflammatory responses → Aspirin, Ibuprofen, Naproxen

Question 36

Imbalanced lipid metabolism can cause health issues. Which are true?
1)