Fatty acid biosynthesis 2 Flashcards
What are the two enzymes involved in fatty acid biosynthesis
- Acetyl-CoA carboxylase
2. Fatty acid synthase
What step is the key regulatory step
- Acetyl-CoA to malonyl-CoA
- Catalysed by acetyl-CoA carboxylase
- Overall rate of fatty acid production is controlled by amount of malonyl-CoA
Describe the structure of acetyl-CoA carboxylase
- Biotin is an essential co-enzyme
- Linked covalently linked to a lysine residue via the ε-amino group
- Mechanism is like that of pyruvate carboxylase
Describe the mechanism of acetyl-CoA carboxylase
- Flexible linking arm which allows attachment of carboxyl group to biotin
- 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
Describe the structure of acetyl-CoA carboxylase in E.Coli
- Multienzyme complex composed of 3 protein components
- biotin carboxyl carrier protein (BCCP)
- biotin carboxylase
- carboxyltransferase - Biotin carboxyl carrier protein is important as the site of attachment
- Other two components are the catalytic units.
Describe the structure of acetyl-CoA carboxylase as an animal enzyme
- A single multifunctional protein.
- Mr of 265 000 (rat liver enzyme)
- Found as a dimer -two identical proteins bound to each other - with one biotin per subunit.
- Non-functional as a dimer and must polymerise to be active
What is the activity of fatty acid synthase
- Multiple activities – multiple active site- different combinations in different species
- Intermediates are bound to the protein
Describe fatty acid synthase in bacteria and plants
- seven activities in seven separate polypeptides- form a complex
- Components can be used
- Different subunits can be attached-used/replaced
Describe fatty acid synthase in yeast
- Seven activities in two separate polypeptides
- One gene encodes protein for 3 parts
- One encodes the other 4 parts
Describe the fatty acid synthase in vertebrates
- seven activities in one large polypeptide
- 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
- More genes in genome in vertebrates
- This means more risk that components would not be in equal proportions - so being all together in one gene means less risk
How is the concentration of intermediates kept high
- Intermediates are not free- not released in solution as concentration would be too low
- Makes it much more efficient- trapped in relatively small area of space
- so effective concentration is very high so much faster rate of reaction
What are two proteins involved in the linkage of substrates to fatty acid synthase
- Beta-ketoacyl ACP synthase
2. Acyl Carrier Protein
Describe the first point of linkage
- Beta-ketoacyl ACP synthase- separate protein in bacteria/plants but domain of single protein in animals
- 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
- A thioester bond forms
- It also holds the longer acyl chain before the condensation with malonyl-ACP.
Describe the second point of linkage
- Acyl Carrier Protein
- the attachment site here is the –SH group of a 4-phosphopantetheine which is in turn linked to a serine side chain of ACP.
- Attached to ACP by phosphate group to OH of serine side chain
- SH group on opposite end attaches to the intermediates
- 4-phosphopantetheine- co-enzyme
Describe 4-phosphopantetheine
- Co-enzyme
- Contains pantothenic acid- vitamin B5
- Relatively long so flexible
- Can access different sites on the fatty acid synthase
What does the shape of the fatty acid synthase of vertebrates look like
- dimer
- Form a cross like structure
- Each protein bent around on itself
Describe how the products are released from the animal enzyme
- Animal enzyme- predominantly released as free fatty acid
2. Palmitate c16:0 (majority released) or a small amount of C18:0 stearate- cleaved away
Describe how the products are released from the yeast enzyme
- Predominantly releases as palmitoyl-CoA
2. The palmitate is transacylated onto CoA from ACP rather than hydrolysed to free fatty acid
Describe how the products are released from the Bacterial/plant enzyme
- About 20% is released as palmitoyl-CoA or palmitoyl-ACP
2. About 70% is released as vaccenate-CoA C18:1 11
What else is required
- Acetyl- CoA
2. NADPH
Why is a transport system for acetyl-CoA required
- Cytosolic acetyl-CoA is the precursor of F.A. synthesis
- Acetyl-CoA is synthesised in the mitochondria- needs to get out into the cytosol
- A transport system is required
How is acetyl-CoA transported into the cytosol
- Acetyl Co-A can’t cross the inner mitochondrial membrane itself
- Acetyl group attaches to oxaloacetate forming citrate- citrate synthase
- Citrate can then pass through the membrane
- The citrate then transforms to oxaloacetate releasing Acetyl-CoA using ATP- citrate lyase
How is oxaloacetate moved back to the mitochondria
- First oxaloacetate forms malate- reduced using NADH and malate dehydrogenase
- Malate can be transported back in and NADH is regenerated
- Or use malic enzyme to convert malate into pyruvate generating NADPH and CO2
- Pyruvate can cross into mitochondria
- Pyruvate carboxylase can convert it to oxaloacetate using energy
How is NADPH produced
- Some is produced from malic enzyme
- The majority is produced by the pentose phosphate pathway
- Glucose-6-phosphate –> ribulose 5-phosphate producing 2 NADPH in the cytosol
Net effect of transport of acetyl-CoA
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
Describe the ratio of NADH/NAD+ in the mitochondria and the result
- High [NADH]/[NAD+]
- This is due to the oxidation of fatty acids, amino acids, pyruvate and acetyl-CoA which occurs in the mitochondria
- This high ratio favours the reduction of oxygen via the respiratory chain
Describe the ratio of NADH/NAD+ in the cytosol and the result
- Low [NADH]/[NAD+] typically 8 x 10-4
2. So NAD+-dependant oxidative catabolism of glucose can occur- Glycolysis can occur
Describe the ratio of NADPH/NADP+ in the cytosol and the result
- High [NADPH]/[NADP+]
- Want glycolysis to occur
- This favours the reductive reactions of F.A. synthesis
Why is NADPH not NADH used in fatty acid biosynthesis
- Use NADPH instead of NADH
- Can maintain different ratio- so high levels of NADPH and low NADH
- So glycolysis can occur at the same time as fatty acid biosynthesis
