Fatty acid biosynthesis 2

Question 1

What are the two enzymes involved in fatty acid biosynthesis

Answer 1

1. Acetyl-CoA carboxylase

2. Fatty acid synthase

Question 2

What step is the key regulatory step

Answer 2

1. Acetyl-CoA to malonyl-CoA
2. Catalysed by acetyl-CoA carboxylase
3. Overall rate of fatty acid production is controlled by amount of malonyl-CoA

Question 3

Describe the structure of acetyl-CoA carboxylase

Answer 3

1. Biotin is an essential co-enzyme
2. Linked covalently linked to a lysine residue via the ε-amino group
3. Mechanism is like that of pyruvate carboxylase

Question 4

Describe the mechanism of acetyl-CoA carboxylase

Answer 4

1. Flexible linking arm which allows attachment of carboxyl group to biotin
2. Because biotin lysine group is flexible it can move, bringing the carboxyl group attached to biotin into a different region with different active site where there is transcarboxylase so the carboxyl group is transferred to acetyl-CoA to make malonyl

Question 5

Describe the structure of acetyl-CoA carboxylase in E.Coli

Answer 5

1. Multienzyme complex composed of 3 protein components
   - biotin carboxyl carrier protein (BCCP)
   - biotin carboxylase
   - carboxyltransferase
2. Biotin carboxyl carrier protein is important as the site of attachment
3. Other two components are the catalytic units.

Question 6

Describe the structure of acetyl-CoA carboxylase as an animal enzyme

Answer 6

1. A single multifunctional protein.
2. Mr of 265 000 (rat liver enzyme)
3. Found as a dimer -two identical proteins bound to each other - with one biotin per subunit.
4. Non-functional as a dimer and must polymerise to be active

Question 7

What is the activity of fatty acid synthase

Answer 7

1. Multiple activities – multiple active site- different combinations in different species
2. Intermediates are bound to the protein

Question 8

Describe fatty acid synthase in bacteria and plants

Answer 8

1. seven activities in seven separate polypeptides- form a complex
2. Components can be used
3. Different subunits can be attached-used/replaced

Question 9

Describe fatty acid synthase in yeast

Answer 9

1. Seven activities in two separate polypeptides
2. One gene encodes protein for 3 parts
3. One encodes the other 4 parts

Question 10

Describe the fatty acid synthase in vertebrates

Answer 10

1. seven activities in one large polypeptide
2. More effective in vertebrates as only want to use the enzyme for fatty acid synthesis- not for making antibiotics etc., so wouldn’t use the separate components like bacteria do
3. More genes in genome in vertebrates
4. This means more risk that components would not be in equal proportions - so being all together in one gene means less risk

Question 11

How is the concentration of intermediates kept high

Answer 11

1. Intermediates are not free- not released in solution as concentration would be too low
2. Makes it much more efficient- trapped in relatively small area of space
3. so effective concentration is very high so much faster rate of reaction

Question 12

What are two proteins involved in the linkage of substrates to fatty acid synthase

Answer 12

1. Beta-ketoacyl ACP synthase

2. Acyl Carrier Protein

Question 13

Describe the first point of linkage

Answer 13

1. Beta-ketoacyl ACP synthase- separate protein in bacteria/plants but domain of single protein in animals
2. The -SH group of a cysteine amino acid residue of this protein acts as an attachment site for the priming group- Acetyl from acetyl-CoA
3. A thioester bond forms
4. It also holds the longer acyl chain before the condensation with malonyl-ACP.

Question 14

Describe the second point of linkage

Answer 14

1. Acyl Carrier Protein
2. the attachment site here is the –SH group of a 4-phosphopantetheine which is in turn linked to a serine side chain of ACP.
3. Attached to ACP by phosphate group to OH of serine side chain
4. SH group on opposite end attaches to the intermediates
5. 4-phosphopantetheine- co-enzyme

Question 15

Describe 4-phosphopantetheine

Answer 15

1. Co-enzyme
2. Contains pantothenic acid- vitamin B5
3. Relatively long so flexible
4. Can access different sites on the fatty acid synthase

Question 16

What does the shape of the fatty acid synthase of vertebrates look like

Answer 16

1. dimer
2. Form a cross like structure
3. Each protein bent around on itself

Question 17

Describe how the products are released from the animal enzyme

Answer 17

1. Animal enzyme- predominantly released as free fatty acid

2. Palmitate c16:0 (majority released) or a small amount of C18:0 stearate- cleaved away

Question 18

Describe how the products are released from the yeast enzyme

Answer 18

1. Predominantly releases as palmitoyl-CoA

2. The palmitate is transacylated onto CoA from ACP rather than hydrolysed to free fatty acid

Question 19

Describe how the products are released from the Bacterial/plant enzyme

Answer 19

1. About 20% is released as palmitoyl-CoA or palmitoyl-ACP

2. About 70% is released as vaccenate-CoA C18:1 11

Question 20

What else is required

Answer 20

1. Acetyl- CoA

2. NADPH

Question 21

Why is a transport system for acetyl-CoA required

Answer 21

1. Cytosolic acetyl-CoA is the precursor of F.A. synthesis
2. Acetyl-CoA is synthesised in the mitochondria- needs to get out into the cytosol
3. A transport system is required

Question 22

How is acetyl-CoA transported into the cytosol

Answer 22

1. Acetyl Co-A can’t cross the inner mitochondrial membrane itself
2. Acetyl group attaches to oxaloacetate forming citrate- citrate synthase
3. Citrate can then pass through the membrane
4. The citrate then transforms to oxaloacetate releasing Acetyl-CoA using ATP- citrate lyase

Question 23

How is oxaloacetate moved back to the mitochondria

Answer 23

1. First oxaloacetate forms malate- reduced using NADH and malate dehydrogenase
2. Malate can be transported back in and NADH is regenerated
3. Or use malic enzyme to convert malate into pyruvate generating NADPH and CO2
4. Pyruvate can cross into mitochondria
5. Pyruvate carboxylase can convert it to oxaloacetate using energy

Question 24

How is NADPH produced

Answer 24

1. Some is produced from malic enzyme
2. The majority is produced by the pentose phosphate pathway
3. Glucose-6-phosphate –> ribulose 5-phosphate producing 2 NADPH in the cytosol

Question 25

Net effect of transport of acetyl-CoA

Answer 25

EITHER when pyruvate is transported
1. Utilisation of 2 ATP with conversion of one NADH to NADPH
2. With transfer of 1 CO2 from the mitochondrion to the cytosol
3. Favoured by high energy conditions
OR when malate is transported
1. Utilisation of 1 ATP 
2. No net change in NAD+/NADH 
3. Favoured by low energy conditions

Question 26

Describe the ratio of NADH/NAD+ in the mitochondria and the result

Answer 26

1. High [NADH]/[NAD+]
2. This is due to the oxidation of fatty acids, amino acids, pyruvate and acetyl-CoA which occurs in the mitochondria
3. This high ratio favours the reduction of oxygen via the respiratory chain

Question 27

Describe the ratio of NADH/NAD+ in the cytosol and the result

Answer 27

1. Low [NADH]/[NAD+] typically 8 x 10-4

2. So NAD+-dependant oxidative catabolism of glucose can occur- Glycolysis can occur

Question 28

Describe the ratio of NADPH/NADP+ in the cytosol and the result

Answer 28

1. High [NADPH]/[NADP+]
2. Want glycolysis to occur
3. This favours the reductive reactions of F.A. synthesis

Question 29

Why is NADPH not NADH used in fatty acid biosynthesis

Answer 29

1. Use NADPH instead of NADH
2. Can maintain different ratio- so high levels of NADPH and low NADH
3. So glycolysis can occur at the same time as fatty acid biosynthesis