β-oxidation of fatty acids Flashcards
What experiment did Knoop carry out
- Published 1904- before radio labelling
- Fed fatty acids labelled at the methyl end with phenyl group to dogs (phenyl group blocks degradation)
- Criticism that phenyl group could be interfering- big artefact
- Some fatty acids had odd number of carbons eg Phenyl propionate
- Some had even numbers of carbons eg Phenyl butyrate
- Examined the excretion products in the urine
7)
What was the result of Knoops experiment
- Discovered with odd chain lengths- benzoate or benzoate derivative in urine
- Even chains- phenyl acetate in urine
- Where there is a β carbon it can be oxidised and then the bond between the α and β carbons can be broken
- Needs to be 2 ch2 groups taken away together
- If enzyme which removes carbon attacks alpha carbon- can do one at a time- benzoate
- If enzymes recognise beta carbon then that explains how two carbons removed at a time
- Beta carbon is c3 from carboxyl end in fatty acid
What are the 3 stages of beta- oxidation
- Activation
- Transport
- Oxidation
Where does activation occur
- In the cytosol on the outer surface of the mitochondrial membrane
What is the enzyme involved in activation
- Acyl-CoA synthetase (thiokinases)
What is the equation for activation
- RCOO- + CoA + ATP ACYL-CoA +AMP + PPi
2. RCOO- = fatty acid
What is the purpose of the activation step
- Ensures subsequent reactions can occur as negative delta G
- Combined hydrolysis of ATP and combination of coA to acyl group- delta G almost 0 so is freely reversible
How is the reaction diven
- Being reversible is not helpful if want to drive reaction to right
- Reaction is driven to the right by removal of a product 3. Pyrophosphatase- breaks pyrophosphate into two phosphates- is ubiquitous found all over mitochondria
- Cleave different phosphodiester bond to form pyrophosphate and adenosine monophosphate
- Hydrolysing pyrophosphate to two inorganic phosphates- removes components from right of reaction and no substrate to go back the other way
- RCOO- + CoA + ATP + H2O –> Acyl-CoA + AMP + 2Pi + 2H+
- Because got to AMP would need to be phosphorylated again twice- used up two ATPs
Why does transportation need to occur
- Fatty acid is in cytosol In surface of mitochondria
2. Acyl groups need to enter mitochondrion to be metabolised (oxidised)
Describe how fatty acyl-CoA is transported across the membrane
- Acyl group attached to coenzyme A outside mitochondria
- Long-chain fatty acyl-CoA cannot directly cross the inner mitochondrial membrane
- Its acyl portion is first transferred to carnitine
- CoA on outside stays outside
- Utilises carnitine as intermediate to carry acyl group through facilitated diffusion through a pore in the inner mitochondrial membrane
- Acyl CoA can easily go through outer- inner is problem
- Acyl carnitine goes in
- Then acyl group is transferred back to CoA releasing carnitine
- Carnitine returns to the cytosol
Who discovered that fatty acids are oxidised in the mitochondrion
- Lehninger and Kennedy
Describe the structure of carnitine
- Has a hydroxyl group
- Acyl group becomes attached in place of hydroxyl and releases Coenzyme A
- Ester linkage rather than thioester linkage
Where does beta-oxidation occur
- In the mitochondrial matrix
Outline the first step of beta-oxidation
- Formation of a trans-alpha,beta double bond through dehydrogenation by acyl-CoA dehydrogenase
Outline the second step of beta-oxidation
- Hydration of the double bond by enoyl-CoA hydratase to form a 3-L-hydroxyacyl-CoA
Outline the third step of beta-oxidation
- NAD+ dependent dehydrogenation of this Beta-hydroxy-acyl-CoA by 3-L-hydroxyacyl-CoA dehydrogenase to form the corresponding beta-ketoacyl-CoA
Outline the fourth step of beta-oxidation
- Calpha-Cbeta cleavage in a thiolysis reaction with CoA as catalysed by Beta-ketoacyl-CoA thiolase (just thiolase)
- forms acetyl CoA and a new acyl-CoA containing two less C atoms than the original
When do the fourth steps of beta-oxidation repeat
- If 4 or more carbons long undergo 4 reactions again
- Keeps repeating
- E.g. Seven cycles to reduce a 16 carbon fatty acid into eight 2 carbon units
Describe the first step of beta-oxidation
- Reduces number of hydrogens present- dehydrogenation reactions
- 3rd carbon from carboxyl end- beta carbon-is attacked
- Why Knoop saw no oxidation with phenyl groups- if beta carbon is carbon in phenyl group it can’t be oxidised
- Double bond forms between beta and alpha carbon
- FAD receives 2 hydrogens
- Produces a trans-delta2-Enoyl-CoA
- Oxidation
Describe the second step of beta-oxidation
- Enoyl-CoA oxidised further by water
- Enzyme- enoyl-CoA hydratase
- Double bond is attacked and broken down
- Hydroxyl group attaches to beta carbon
- Alpha carbon regains a hydrogen
- Forms beta-3-hydroxy-acyl-CoA
- Hydration
Describe the third step of beta-oxidation
- Removal of 2 hydrogens
- Carrier- NAD
- Start with hydroxy acyl Co-A
- Enzyme hydroxy acyl Co-A dehydrogenase
- Hydrogens removed from hydroxyl group and beta carbon
- Forms a ketone group- Beta-ketoacyl-CoA
- Oxidation
Describe the fourth step of beta-oxidation
- Enzyme- acyl-CoA-acetyltransferase (thiolase)
- CH2CO group is removed and forms acetyl-CoA
- The C=O of the remaining molecule reattaches to CoA forming Acetyl-CoA -Most will go into citric acid cycle
- The acyl-CoA now has 2 less carbons
- These 4 steps are repeated each time removing 2 carbons
What is the overall equation for beta oxidation of a fatty acid and give an example using palmitoyl-CoA
- Cn-ACYL-CoA + FAD + NAD+ + H2O + CoA –> Cn-2-ACYL-CoA + FADH2 + NADH + H+ + ACETYL-CoA
- E.g. Palmitoyl-CoA + 7 FAD + 7 NAD+ + 7 H2O + 7 CoA –> 8 acetyl-CoA + 7 FADH2 + 7 NADH + 7H+
- 7 repeats of 4 step process
What happens to the acetyl-CoA produced
- Enters the TCA cycle
- Made into ketone bodies
- Most goes into the TCA cycle unless there is a shortage of carbohydrate/a lot of fatty acid metabolism
What is the overall equation for the citric acid cycle
- Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2H2O ->CO2 + 3 NADH + 3H+ + FADH2 + GTP + CoA
How much NADH, FADH2 and GTP molecules are formed from the acetyl-CoA produced from oxidation of palmitate in TCA
- As there are eight acetyl-CoA from palmitate the total production in TCA
- 8 x 3 = 24 NADH + H+
- 8 x 1 = 8 FADH2
- 8 x 1 = 8 GTP
How much NADH, FADH2 and GTP is produced from the beta oxidation of palmitate
- The steps of β–oxidation produce some FADH2 and NADH + H+
- For palmitate that is:
- 7 FADH2
- 7 NADH + H+
- No GTP
What is the total production of NADH, FADH2 and GTP from the beta oxidation of palmitate including TCA
- 24 + 7 = 31 NADH + H+
- 8 + 7 = 15 FADH2
- 8 = 8 GTP
What is the relation between GTP and ATP
- GTP + ADP–>ATP + GDP
2. So 8 GTP make 8 ATP
How much ATP does NADH + H+ and FADH2 produce in oxidative phosphorylation
- NADH + H+ - theoretical- 3.0 but measured 2.5
- FADH2 - theoretical- 2.0 but measured 1.5
- Either can calculate theoretical amount- more comparable but may not be strictly accurate- mainly stick with theoretical amount
How much ATP is generated in the Beta-oxidation of palmitate including the TCA
- 31 NADH+H+ * 3 = 93 ATP [77.5]
- 15 FADH2 * 2= 30 ATP [22.5]
- 8GTP = 8 ATP
- Overall = 131 ATP [108 ATP]
- BUT- this is just from palmitate-CoA need to remember ATP used to convert palmitate to palmitate CoA
- Used 1 atp but took 2 phosphates from it = 2 ATP
- So OVERALL = 129 ATP [106ATP]
How much is the energy captured per mole of ATP
- Energy captured is 129 times the Free energy of hydrolysis of one phosphate from ATP
- 129 x -31kJ/mole = -3999kJ/mole
What is the efficiency of beta oxidation of palmitate
- Standard Free energy of oxidation of palmitate is -9790kJ/mole
- Calculated by burning it in calorimeter
- Proportion of energy captured = -3999 / -9790= ~40%
What is the yield of ATP per carbons oxidised to CO2
- No. of ATP/ No. of carbons = 129 / 16 = ~8.2
- Equivalent figure for glucose is ~6.3
- Fats can generate more atp per carbon than carbohydrates
Describe storage of energy in fats compared to carbohydrates
- Fats aren’t stored with water- less volume taken up than with carbohydrates
Describe need for oxaloacetate
- Oxaloacetate is needed for entry of acetyl-CoA into the TCA cycle
- Not always present in large enough quantities e.g. during fasting
- TCA not 100% efficient- not all molecules come back as oxaloacetate
- Need anaplerotic reactions- regenerate or reproduce oxaloacetate so can continue around
What is the ketotic state
- Ketotic state- some acetyl CoA can’t go into TCA cycle
- So have to form ketone bodies
- When not enough carbohydrate
What happens when Acetyl-CoA cannot enter TCA cycle
- When it cannot enter the TCA cycle: Acetyl-CoA –>acetoacetate + D-3-hydroxybutyrate
- Acetoacetate + D-3-hydroxybutyrate- both ketone bodies
Where does the formation of ketone bodies mainly occur
- Mostly occurs in liver as undergoes gluconeogenesis
- Oxaloacetate is being withdrawn in those cells for synthesis of glucose- gluconeogenesis
- Key enzymes found in liver
What are the properties of ketone bodies
- energy rich
- Water soluble – so easily transported in the blood- unlike fats
- Heart and renal cortex are major users of ketone bodies- use them as preferred fuel
How are ketone bodies formed
- Reaction is reverse of last step of beta oxidation
- 2 acetyl-CoA react with thiolase producing acetoacetyl-CoA
- In mitochondria
- Reforms acetoacetyl-CoA or never forms acetyl-CoA from acetoacetyl-CoA in beta oxidation
- SHORTAGE of Co-A also keeps it as acetoacetyl CoA
How is the build-up of acetoacetyl-CoA prevented
- HMG-CoA synthase takes another acetyl-CoA and combines with acetoacetyl-CoA to form hydroxy-methylglutaryl-CoA
- HMG-CoA also used to synthesis cholesterol- normal function
- But liver also has HMG-CoA lyase which removes a acetyl CoA forming acetoacetate
What happens to the acetoacetate
- Can diffuse through mitochondrial membrane- water soluble
- Can be reduced by H+ +NADH- Concentration gradient to form beta-hydroxybutyrate by beta-hydroxybutyrate dehydrogenase
- Acetoacetate decarboxylase- take carboxyl carbon (CO2) from acetoacetate to give acetone
How are ketone bodies used
- Beta-hydroxybutyrate can be used to produce acetoacetate- reverse of other reaction
- Acetoacetate can be turned to acetoacetyl-CoA using Succinyl-CoA to succinate
- Formation of Succinyl-CoA requires energy input
- Acetoacetyl-CoA converted to 2-acetyl-CoA by thiolase these enter TCA cycle to give more ATP
- Utilisation of ketone bodies reverse of formation
