Lecture 30 - The final oxidation: The citric acid cycle

Question 1

Citric acid cycle

Answer 1

This is cyclical pathway that the acetyl-CoA molecules generated from glycolysis and beta oxidation go. Acetyl-CoA enters this pathway (getting it started), and as acetyl-CoA is used up by CAC, some NADH+H+, FADH2 and ATP are generated. CO2 is released during this pathway too - this also contributes to the carbon dioxide that we exhale when our bodies are doing work

Question 2

Where does the citric acid cycle occur?

Answer 2

Occurs in the mitochondria
All but one of the enzymes is in the mitochondrial matrix (succinate dehydrogenase (associated complex II) which is also involved in the citric acid cycle)

Question 3

Other names for the citric acid cycle

Answer 3

Krebs cycle

TCA (tricarboxylic acid) cycle

Question 4

Citric acid cycle summary

Answer 4

Cycle - starts and finishes with the same molecule (oxaloacetate - regenerated in each cycle, four carbon acceptor molecule)

Carbon enters and leaves

• 2C in acetyl-CoA in
• 2C as CO2 out (oxidation complete)

Captures energy:
1 ATP
3 NADH and 1 FADH2

Question 5

The citric acid cycle is arranged into two parts….

Answer 5

1 - the release of carbon
2- Regeneration of oxaloacetate, the starting molecule

Energy is captured in both

Question 6

Condensation of acetyl-CoA with oxaloacetate

Answer 6

2 carbon entering the CC as acetyl-CoA is attached to 4 carbon oxaloacetate to produce 6 carbon citrate and CoA-SH
Energy for the reaction comes form the hydrolysis of CoA from acetyl-CoA
Because the CAC is a ‘cycle’ these two carbons need to be removed
OH from water makes the citrate and H from water makes CoA-SH

Negative delta G so the reaction is energetically favourable

Question 7

Isomerisation of citrate

Answer 7

Rearrangement of citrate to isocitrate makes the molecule susceptible to decarboxylation
Both steps are catalysed by aconite

Question 8

Targeting the CAC to kill…

Answer 8

Both nature and man have targeted the CAC as a way of killing animals
Fluroacetate is found in the leaves of certain plants and this is a molecule that can inhibit the citric acid cycle
The poison 1080 is sodium fluroacetate.
Fluroacetate is metabolised to fluorocitrate

Flucoacetate has CoA-SH added to it to produce fluoroacetyl-CoA and then water is added and through citrate synthase fluorocitrate is formed. Fluorocitrate inhibits the acontiase enzyme due to the presence of a fluorine instead of a hydrogen

Question 9

Fluroacetate vs no fluoroacetate

Answer 9

Flucoacetate has CoA-SH added to it to produce fluoroacetyl-CoA and then water is added and through citrate synthase fluorocitrate is formed. Fluorocitrate inhibits the acontiase enzyme due to the presence of a fluorine instead of a hydrogen

No fluoroacetate …
Oxaloacetate has water added and with citrate synthase gets turned into citrate. Rearrangement of the citrate to isocitrate makes the molecule susceptible to decarboxylation. Both steps are catalysed by aconitase (citrate to cis-aconitate to isocitrate)

Question 10

Key reaction … the removal of the first carbon

Answer 10

An oxidative decarboxylation
Reaction occurs in two steps - oxidation then the decarboxylation
Energy is captured in NADH

The enzyme involved is called isocitrate dehydrogenase (isocitrate to oxalosuccinate (enzyme bound) to alpha-ketoglutarate)

Question 11

Key reaction … the removal of the second carbon

Answer 11

A second oxidative decarboxylation
Very similar to the pyruvate dehydrogenase reaction
ENergy captured in NADH

The enzyme involved is called alpha-ketoglutarate dehydrogenase (alpha-ketoglutarate to succinyl-CoA)

Question 12

Summary of the first part of the citric acid cycle

Answer 12

Two carbons are removed
Energy is conserved as NADH
A four C molecule has been regenerated (succinyl-CoA)

The rest of the cycle is concerned with regenerating oxaloacetate

Question 13

Key reaction …. Succinyl-CoA to succinate

Answer 13

The conversion of succinyl-CoA to succinate is like the activation of fatty acids in reverse
The removal of the CoA releases enough energy to drive the synthesis of GTP
GTP is the energy equivalent of ADP (GTP + ADP GDP + ATP)
The 3rd substrate level phosphorylation in the pathways

Question 14

Substrate level phosphorylation

Answer 14

The direct use of energy from a substrate molecule to drive the synthesis of ATP (or equivalent)
The phosphate does not have to come from the substrate, the energy is coming from the cleavage of the bond of the CoA from the succinyl-CoA to succinate reaction

Question 15

Succinate to oxaloacetate

Answer 15

Succinate to fumarate via succinate dehydrogenase (oxidation with FAD to FADH2) - single bond to double bond, losing hydrogen and donating them to FAD which is then reduced to FADH2
Fumarate to malate via fumarate (hydration)
Malate to oxaloacetate via malate dehydrogenase (oxidation with NAD+ to NADH + H+) - reduction of NAD+ to NADH + H+

The reactions used to convert succinate to oxaloacetate are very similar to beta oxidation

Question 16

Key reaction … a shared reaction

Answer 16

Succinate dehydrogenase (SDH) is located in the inner mitochondrial membrane 
SDH used FAD as a coenzyme - flavin coenzymes are tightly bound to the protein with which they interact (flavoproteins) 
SDH is part of the CAC where FAD is reduced, so needs to also be in the ETC (associated with complex II) to oxidise FADH2 back to FAD

Succinate to fumarate is what succinate dehydrogenase is used for

Question 17

Overall reaction for the CAC

Answer 17

acetyl-CoA + 3NAD+ + FAD + 2H2O + GDP + Pi -> 2CO2 + CoASH + 3NADH + 3H+ + FADH2 + GTP

Question 18

Gibbs energy of the overall citric acid cycle

Answer 18

-44.3 kJ/mol

Negative therefore this reaction is energetically favourable