Lipid Oxidation and Biosynthesis

Question 1

What happens to stored triglycerides in the fasting state?

Answer 1

They are released from adipose tissue by being broken down (lipolysis) using a hormone sensitive lipase
When in the bloodstream they bind to serum albumin in order to maintain solubility

Question 2

Where is a major site of fatty acid oxidation?

Answer 2

Muscle, however fatty acids are the usual oxidative substrate of most tissues in the unfed state

Fatty acids cannot cross the blood brain barrier - therefore limited stores of glucose are only used by the brain and for high intensity activity

Question 3

How do we release fatty acids from triacylglycerols?

Answer 3

Lipolysis
Triacylglycerol -> Diacylglycerol (releasing fatty acids)
Diacylglycerol -> monoacylglycerol (releasing fatty acids)
Monoacylglycerol -> Glycerol (releasing fatty acids)

Question 4

What enzymes are used in lipolysis?

Answer 4

1 – Adipose triglyceride lipase (ATGL) + hormone sensitive lipase (HSL)
2 – HSL
3 – HSL + monoacylglycerol lipase

Question 5

What happens to a fatty acid once it has been released from the triaclyglycerides?

Answer 5

Once a fatty acid is released it is acylated with ATP and then attaches to CoA (activated before oxidation)
Fatty acid + ATP + CoA -> acyl-CoA + PPi + AMP
then transported from cytosol to mitochondrion by carnitine (where FA are oxidised)

Question 6

What enzymes are used in order to activate fatty acids? Intermediate?

Answer 6

Thiokinases (acyl-CoA synthetases)
They are associated with either the ER or the outer mitochondrial membrane

Acyladenylate mixed anhydride intermediate
It gets attacked by the sulfhydryl group of CoA to form the thioester product

Question 7

How does carnite transport fatty acids across the inner mitochondrial membrane?

Answer 7

The acyl group of acyl-CoA is tranferred to carnitine using carnite palmitoyl transferase
Forming acyl-carnitine and releasing CoA into the cytosolic pool
The acyl-carnitine is transported into the mitochondrial matix using a carnitine carrier protein
The acyl group is transferred to a CoA molecule in the mitochondiral pool, leaving carnitine to return to the cytosol

Question 8

How does normal fatty acid oxidation proceed?

Answer 8

1. Oxidation by flavoenzyme acyl-CoA dehydrogenase (AD) forming a trans, beta C=C
2. Hydration of C=C by enoyl-CoA hydratase (EH) to 3-L-hydroxyacyl-CoA
3. Oxidation by 3-L-hydroxyacyl-CoA (HAD) to β-ketoacyl-CoA
4. C-C cleavage (claisen ester cleavage) by CoASH catalysed by β-ketoacyl-CoA thiolase (KT) to fatty acyl-CoA (2C shorter) and acetyl CoA

Question 9

How is FAD maintained in step 1 of normal fatty acid synthesis?

Answer 9

FADH2 is re-oxidised through a series of electron transfer reactions

1. Electron-transfer flavoprotein (ETF) transfers an electron pair from FADH2 to ETF:ubiquinone oxidoreductase
2. ETF:ubiquitinone oxidoreductase reduces coenzyme Q (CoQ) to transfer an electron pair to the mitochondrial electron-transport chain
3. Reduction of O2 → H2O ⇒ ~1.5 ATP per electron transferred

Question 10

What is the result of oxidising normal fatty acid chains?

Answer 10

We are only producing reduced cofactors NADH and FADH2, which feeds into the electron transport chain
No ATP is formed directly but from each fat molecule = 106 ATP
Unsaturated fatty acids require additional steps to converts cis double bonds to trans form
Therefore it needs additional enzymes

Question 11

What additional enzymes are needed for oxidation of unsaturated fatty acids e.g. linoleic acid?

Answer 11

Enoyl-CoA isomerase converts cis double bond to trans as cis can’t act as a substrate

Enzymes are needed – first to reduce the double bond - second to ‘shift’ it to the right pair of C atoms (to prevent inhibition of hydratase)

The 3,2- isomerase has another substrate which could prevent 20% of the fatty acid being oxidised
So 3,5-2,4 dienoyl-CoA isomerase converts the compound into a substrate for the 2,4 dienoyl-CoA reductase

Question 12

What are odd-chain fatty acids?

Answer 12

Fatty acids with non-even numbers of C-atoms
We acquire them from diet
Plants and marine organisms can synthesise fatty acids with odd numbers

Question 13

How are odd-chain fatty acids oxidised?

Answer 13

The final round of oxidation of these fatty acids produces propionyl-CoA

1. Propionyl-CoA is converted to (S)-methylmalonyl-CoA, catalysed by propionyl-CoA carboxylase, ATP and a biotin prosthetic group
2. (S)-methylmalonyl-CoA is converted to (R)-methylmalonyl-CoA by methylmalonyl-CoA racemase
3. (R)-methylmalonyl-CoA is converted to succinyl-CoA by methylmalonyl-CoA mutase

Succinyl-CoA can now enter the TCA cycle

Question 14

What prosthetic group does methylmalonyl-CoA mutase have?

Answer 14

5′-deoxyadenosylcobalamin

The structure involves a similar structure to the heme group but contains Cobalt in the middle (no Fe)
Requires a vitamin B12 as a cofactor
It has a unique a/b barrel

Question 15

What is the mechanism of methylmalonyl-CoA mutase?

Answer 15

1. Homolytic cleavage of the C—Co(III) bond yields a 5′-deoxyadenosyl radical and cobalamin
2. The 5′-deoxyadenosyl radical takes a hydrogen atom from methylmalonyl-CoA = methylmalonyl-CoA radical
3. Carbon skeleton rearrangement = a succinyl-CoA radical via a proposed cyclopropyloxy radical intermediate
4. The succinyl-CoA radical takes a hydrogen atom from 5′-deoxyadenosine to regenerate the 5′-deoxyadenosyl radical
5. The release of succinyl-CoA re-forms the coenzyme

Question 16

How does the product of odd-chain fatty acid oxidation enter the TCA cycle?

Answer 16

Succinyl-CoA can’t enter directly
It is converted to malate (reactions 5-7 of TCA)
Then it is transported to the cytosol before undergoing oxidative decarboxylation to pyruvate and CO2, carried out by malic enzyme

Question 17

What else can also oxidise fatty acids?

Answer 17

Peroxisomes

In plants, fatty acid oxidation occurs totally in peroxisomes and glyoxysomes

Question 18

How does peroxisomal beta-oxidation occur?

Answer 18

Peroxisomal β-oxidation shortens fatty acids with chains >22 C atoms – then full degradation in mitochondria
Three enzymes are required:
1. Acyl-CoA oxidase:
fatty acyl-CoA + O2 → trans-Δ2-enoyl-CoA + H2O2
It also uses FAD as a cofactor

2. Peroxisomal enoyl-CoA hydratase:
   C=C bond hydration and dehydrogenation
3. Peroxisomal thiolase:
   removal of acetyl-CoA

Question 19

What are some peroxisome diseases?

Answer 19

Zellweger syndrome - caused by reduction/absence of functional peroxisomes. Leads to neurological problems, vision and hearing loss
X-ALD - defects in the peroxisomal transferase for long chain fatty acids. Leads to adrenal gland dailure and destroys the myelin sheath (axons)

Question 20

What can also happen to acetyl-CoA after being produced in FA oxidation?

Answer 20

If acetyl-CoA is in abundance it can be further oxidised to ketone bodies in the liver to be used as fuel by other tissues - ketogenesis

Question 21

What are ketone bodies used for?

Answer 21

Important for metabolic fuels of peripheral tissues, mainly in the heart and skeletal muscles
Small water soluble ketone bodies are used by the brain during starvation

Question 22

How does acetoacetate (ketone body) form?

Answer 22

1. Two molecules of acetyl-CoA combine to acetoacetyl-CoA, catalysed by thiolase (acetyl-CoA acetyltransferase)
2. Condensation of acetoacyl-CoA and another aceytl-CoA = HMG-CoA, this uses HMG-CoA synthase
3. HMG-CoAis degraded to acetoacetate and acetly-CoA, using HMG-CoA lyase
   This is a mixed aldol-claisen ester cleavage reaction

Question 23

What other ketone bodies can be derived from acetoacetate?

Answer 23

Acetone - happens spontaneously and releases CO2
(ketosis - where acetoacetate is produced faster than metabolised = breath smells of acetone)

3-hydroxy butyrate - uses 3-hydroxy butyrate dehydrogenase and NADH

Question 24

What is fatty acid biosynthesis?

Answer 24

The reverse of oxidation of fatty acids
Excess acetyl CoA can be used to synthesise fatty acids for conversion to triacylglycerols for storage
However, acetyl CoA cannot cross the inner mitochondrial membrane - therefore it uses the tricarboxylate transport system

Question 25

How does the tricarboxylate transport system work?

Answer 25

1. Mitochondrial acetyl CoA is converted to citrate by reaction with oxaloacetate (using citrate synthase)
2. Citrate crosses the inner mitochondrial membrane via the transport system
3. In the cytosol citrate is broken down to form acetyl CoA and oxaloacetate (using ATP-citrate lysase)
4. Oxaloacetate is converted to malate and then pyruvate which can cross back into the mitochondria (using malate dehydrogenase and malic enzyme)
5. In the mitochondria, pyruvate is converted to oxaloacetate - repeat

Question 26

What is step 1 of fatty acid biosynthesis?

Answer 26

Malonyl CoA is synthesised:
Biotinyl + HCO3- + ATP produces caboxybiotinyl-enzyme
Then add acetyl-CoA and you produce malonyl-CoA

This reaction is cataylsed by acetyl-CoA carboxylase (ACC1 form in adipose tissue) - a biotin-dependent enzyme
This is a rate-controlling step

Question 27

What is step 2 of fatty acid biosynthesis?

Answer 27

Synthase is ‘loaded’ with the precursors for the condensation reaction

Malonyl/-acetyl-CoA-ACP transacylase (MAT) catalyzes two similar reactions at a single active site:
An acetyl group originally linked as a thioester in acetyl-CoA is transferred to ACP
A malonyl group is transferred from malonyl-CoA to ACP
(there is a phosphopantetheine group in both ACP and CoA)

Question 28

What is step 3 of fatty acid biosynthesis?

Answer 28

b-ketoacyl-ACP synthase (KS) transfers the acetyl group from ACP onto a cystine residue on KS
Malonyl ACP is decarboxylated – this step drives the reaction
Resulting carbanion attacks the thioester bond
Acetoacetyl ACP is formed
After a few reductions and dehydrogenases (7 cycles) we can work to palmitate for example

Question 29

What are the differences in biosynthesis from oxidation?

Answer 29

Biosynthesis is in the cytoplasm not the mitochondrion
ACP is the acyl carrier not CoA
NADPH = electron donor not FAD or NAD
C2 unit donor is malonyl-CoA not acetyl-CoA

Question 30

Describe the enzyme fatty acid synthase?

Answer 30

Catalyses 7 different reaction at 6 different active sites = efficient
It is a dimer
The long phosphopantethine prosthetic group on ACP moves the growing fatty acid chain between active sites during the reaction
ACP can reach all active sites – but these are not in order of the reactions in the cycle
The two subunits form an asymmetric X, that associate via an extensive interface

Question 31

What happens with an rapid expression of fatty acid synthase?

Answer 31

Overexpression of the enzyme can lead to cancerous cells

Therefore fatty acid synthase inhibitors promote apoptosis in the cancer cells

Question 32

How can fatty acids be modified?

Answer 32

They can be elongated by elongases or desaturated by desaturases

Elongated - can take place in the mitochondria or ER
The mitochondrion uses the reversal of fatty acid oxidation but the final step uses NADPH insteas of FADH2
The ER involves addition of malonyl CoA

Desaturated - terminal desaturases can insert double bonds at Δ9, Δ6, Δ5, Δ4 positions

Question 33

How are essential unsaturated fatty acids acquired in animals?

Answer 33

Via diet
e.g. linoleic acid and linolenic acid

As the double bond can’t be formed beyond C9 but linoleic acid for example is at C12