Fatty acids (synthesis and breakdown) Flashcards
roles of fatty acids
component of complex lipids (e.g. triacylglycerols)
component of cell membranes
can act as signalling molecules at low concentration
what are essential fatty acid
fatty acids that humans can’t synthesise, so must be consumed
normally unsaturated fatty acids
what is fatty acid turnover
dynamic process of fatty acid synthesis, storage and breakdown
makes up 8% of body’s basal oxygen consumption
how to work out whether fatty acid synthesis occurs in cytoplasm or nucleus
- homogenisation
- centrifugation (splits to pellet and soluble protein)
adding reaction components to either the pellet or soluble protein solution and seeing where there is activity
Will be in cytosol
how to work out which proteins in cytosol are involved in fatty acid biosynthesis
- homogenisation
- centrifugation
- precipitation:
add small amount of ammonium sulphate (large water hydration shell)
least soluble proteins ppt (salting out) - centrifuge - separates from more soluble proteins still in solution
- repeat - results in sub fractions each with different proteins
what happens when you test each individual sub fraction for fatty acid biosynthetic activity
no single fraction produces activity
combining 2 fractions (one must contain biotin) and biosynthetic activity will always be produced
stage 1 of fatty acid biosynthesis
carboxylation of acetyl coA - irreversible 2 stage process
Requires biotin to move the CO2
Reactants: ATP, acetyl CoA, HCO3-
Products: malonyl CoA, water, ADP + Pi
enzyme: acetyl CoA carboxylase
step 1 of stage 1 (carboxylation of CoA) of fatty acid biosynthesis
biotin + HCO3- => biotin-CO2
converts ATP -> ADP + Pi (provides the energy required for the reaction)
Catalysed in the biotin carboxylase domain
acetyl coA carboxylase enzyme
catalyses carboxylation of acetyl CoA to form malonyl CoA
2 step process
2 domains (one for each step)
Biotin carboxylase domain
Transcarboxylase domain
role of biotin in stage 1 of fatty acid biosynthesis
carries CO2 from acetyl CoA to malonyl coA by forming a carboxybiotin intermediate
step 2 of stage 1 (carboxylation of CoA) of fatty acid biosynthesis
Biotin-CO2 + acetyl CoA => malonyl CoA + biotin
Occurs in transcarboxylase domain of enzyme
role of acetyl CoA and malonyl CoA in FA biosynthesis
acetyl CoA - primes reaction (2 carbons become the methyl terminus of FA chain)
malonyl CoA - extends the chain after carboxyl carbon (each provides 2 carbons)
overall reaction for stage 2 of fatty acid biosynthesis
include enzyme used
acetyl CoA + 7 malonyl CoA + 14NADPH + 14H+ => palmitate (C16) + 7CO2 + 8CoA + 6H2O + 14NADP+
catalysed by fatty acid synthase
takes part in 7 steps
what were the results of the experiment carried out by Frans Knoop to investigate breakdown of fatty acids?
- investigated a range of fatty acids that were labelled with a phenyl group at their methyl end (this blocks degredation)
- found that FA with an odd carbon number formed benzoate
- FA with even carbons formed phenyl acetate
why did frans knoop get these results
odd number => benzoate
even number => phenyl acetate
beta carbon is attacked during oxidation (3rd one from carboxyl end)
with odd chains, the beta carbon is available to be oxidised to a carboxyl group
with even chains, beta carbon part of the phenyl group and can’t be attacked
3 steps of beta oxidation
- activation (add CoA to FA forming acyl CoA
- transport of acyl-coA to MT matrix
- oxidation of FA
Step 1 of beta oxidation of FA (activation)
FA gets attached to CoA, forming acyl CoA
RCOO- + CoA + ATP <=> Acyl-CoA + AMP + PPi
remove PPi to shift eqm to right (since delta G is close to 0)
phosphatase cleaves into 2 phosphates in a hydrolysis reaction
PPi + H2O => 2Pi + 2H+
step2 of beta oxidation of FA?
Transport acyl-CoA to mitochondrial matrix
outer mem = permeable
inner mem =use carnitine shuffle
outside matrix:
carnitine + acyl CoA => CoA + acyl-carnitine
Catalysed by carnitine acyltransferase I
inside matrix
acyl-carnitine + CoA => carnitine + acyl CoA
enzyme =carnitine acyltransferase II
why is carnitine sometimes taken as a supplement when it is synthesised by humans?
increases rate of fat into mitochondria
utilise more for energy
weight loss
limited evidence
step 3 of beta oxidation of fatty acids?
4 step process which removes a 2 carbon unit
repeats if the FA is still 4 or more carbons long
what step is regulated in fatty acid biosynthesis and why is this important?
first step, catalysed by acetyl-coA carboxylase is regulated
means all the other enzymes in the pathway can occur at the rate substrate is produced
prevents a build up of intermediates in the pathway
3 domains of acetyl-CoA carboxylase
2 enzymatic domains - biotin carboxylase domain and transcarboxylase domain
1 structural domain - biotin carrier protein (biotin is attached)
how does the acetyl-CoA carboxylase enzyme different in bacteria and vertebrates?
in bacteria (e. coli), there are 3 separate genes controlled by an operon > produce 3 separate proteins
in humans - single large gene, producing single polypeptide with 3 components
how does acetyl-CoA exist in nature?
exists as a dimer, forming a cross-like structure
one biotin per subunit of the dimer
(each subunit has all 3 domains)
however, it is non functional as a dimer and must polymerase to be active
compare structure of fatty acid synthase in plants and animals?
plants - 7 different proteins that come together, but not controlled by operons (must have coordinated regulation to ensure a 1:1 ratio)
animals - seven activities within one large polypeptide
why do plants have 7 different proteins if they dont have any operons
separate proteins enables assembly in different ways
can leave out some components to increase range of fatty acids that can be made (e.g differing numbers of ketone groups and double bonds = saturated and unsaturated)
how are intermediates linked to the beta-ketoacyl synthase domain of FA synthase?
within the KS domain, there is a cysteine residue
-SH on cysteine residue holds intermediates via a thioester bond
(e.g. attachment of acetyl group/ acyl chain before condensation with malonyl-ACP)
how are intermediates linked to the acyl carrier protein domain of FA synthase
prosthetic group of ACP is 4’ phosphopantetheine, which has a -SH bond
-SH group attaches to intermediates via a thioester bond
4’ phosphopantetheine is linked to ACP via ester bond
how are fatty acids released from FA synthase?
thioesterase - hydrolyses thioester bond between the growing chain and 4’phosphopantetheine
how is the fatty acid released in animals, yeast and bacteria/ plants?
animals - released as palmitate (C16:0) or stearate (C18:0)
yeast - released as palmitoyl-CoA (palmitate is transacylated onto CoA)
Bacteria/ plants - 20% palmitoyl CoA/ palmitoyl ACP and 70% vaccenate-CoA (C18:1 delta 11)
2 other major requirements for fatty acid biosynthesis?
- requires acetyl-CoA in the cytosol (must be transported, as no process produces it here)
- requires a supply of NADPH (to reduce the acyl chain in step 4 of FA synthase reaction)
In what conditions are the 2 transport pathways for CoA used?
Pathway via pyruvate - high energy (when lots of ATP is being produced)
Uses 2 ATP
Main aim for FA biosynthesis is storage
Pathway via malate - low energy
Uses 1 ATP
how is supply of NADPH (for fatty acid biosynthesis) provided?
Transport of acetyl-CoA only produces 1 NADPH (if high energy, and no NADPH is low energy path taken)
Therefore, 2 NADPH also produced via the pentose phosphate pathway
This converts G6P into ribulose-5-phosphate through a rearrangement of carbon atoms)
why is NADPH used as the reducing agent and not NADH?
glycolysis also occurs in the cytosol -
In cytosol, there is low NADH: NAD ratio to enable glycolysis to occur (as it produces NADH so must be little to start)
Instead, cytosol has high NADPH: NADP ratio, ensuring lots of NADPH for FA biosynthesis to occur
4 steps of oxidation (stage 3 of beta oxidation of fatty acids)
- oxidation
- hydration
- oxidation
- thiolytic cleavage
(process in more detail on summary page)
