Lecture 3 - The Citric Acid Cycle

Question 1

How is acetly CoA formed?

Answer 1

Acetyl CoA is formed from pyruvate when it is oxidatively decarboxylated by the pyruvate dehydrogenase complex to form Acetyl CoA.

Pyruvate + CoA + NAD+ –> acetyl CoA + CO2 + NADH + H+

Acetyl CoA is the fuel for the citric acid cycle.

Question 2

Give an overview of the citric acid cycle.

Answer 2

Step 1 - Citrate is formed from oxaloacetate and acetyl coenzyme A
Step 2 - Citrate is Isomerised into Isocitrate
Step 3 - Isocitrate is oxidised and decarboxylated to alpha - ketoglutarate
Step 4 - Succinyl coenzyme A is formed by the oxidative decarboxylation of alpha-ketoglutarate
Step 5 - Succinyl CoA is used to generate Succinate which has a high phosphoryl transfer potential
Step 6 - Oxaloacetate is regenerated by the oxidation of succinate

Question 3

Describe step 1 of the citric acid cycle.

Answer 3

Step 1 - Citrate is formed from oxaloacetate and acetyl coenzyme A

This reaction is catalysed by the enzyme citrate synthase.

• Oxaloacetate combines with acetyl CoA to form citryl CoA
  The Citryl CoA is an energy rich molecule due to the thioester bond that originated in the acetyl CoA. The hydrolysis of this molecule to Citrate and CoA drives the overall reaction to synthesise Citrate.
• The Citrate synthase is composed of two identical subunits. When oxaloacetate binds to the enzyme (Open form) it induces a major structural change which creates a binding site for acetyl CoA (closed form). The reaction is catalysed as the enzyme forces the substrates into close proximity orientating them and polarising certain bonds. An enol intermediate is formed.
  The enzyme ensures the hydrolysis of Citryl CoA but not acetyl CoA which prevents the wasteful hydrolysis of acetyl CoA

Question 4

Describe step 2 of the citric acid cycle.

Answer 4

Step 2 - Citrate is Isomerised into Isocitrate
Catalysed by aconitase

In order to undergo oxidative decarboxylation the citrate needs to be isomerized into isocitrate as the hydroxyl group is not in the right place in the citrate molecule.

The isomerisation of citrate is accomplished by a dehydration step followed by a rehydration step forming cis-aconitate as an intermediate

Question 5

Describe step 3 of the citric acid cycle.

Answer 5

Step 3 - Isocitrate is oxidised and decarboxylated to alpha - ketoglutarate
Catalysed by isocitrate dehydrogenase

Isocitrate is oxidised to the intermediate oxalosuccinate. The hydrogen which is lost reduces NAD+ to NADH

The oxalosuccinate is decarboxylated (loses as CO2) to form α-ketoglutarate

Question 6

Describe step 4 of the citric acid cycle.

Answer 6

Step 4 - Succinyl coenzyme A is formed by the oxidative decarboxylation of alpha-ketoglutarate

Catalysed by the α-ketoglutarate dehydrogenase complex an assembly of three different kinds of enzymes. Deficiencies in thiamine will also induce low activities of α-ketoglutarate dehydrogenase which can lead to beriberi and is associated with amnesia.

The α-ketoglutarate is decarboxylated
It forms a thioester linkage with CoA
The overall reaction forms Succinyl CoA, CO2 and NADH

Question 7

Describe step 5 of the citric acid cycle.

Answer 7

Step 5 - Succinyl CoA is used to generate Succinate which has a high phosphoryl transfer potential
Catalysed by succinyl CoA synthetase

The cleavage of the thioester bond of succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate usually ADP or GDP.

a. Coenzyme A is displaced by orthophosphate. This generates succinyl phosphate which is energy rich
b. A histidine residue acts as a moving arm that detaches the phosphoryl group
c. It then moves over to a bound ADP
The phosphate is transferred to form ATP

Question 8

Describe step 6 of the citric acid cycle.

Answer 8

Step 6 - Oxaloacetate is regenerated by the oxidation of succinate

Catalysed by:
> Succinate dehydrogenase (Bound to the inner membrane of the mitochondria. FAD is bound to the enzyme)
> Fumarase
> Malate dehydrogenase - Catalyses the reversible oxidation of malate with the equilibrium towards malate however the reaction is driven by the utilisation of the products.

A methylene group is converted into a carbonyl group in three steps: an oxidation, a hydration and a second oxidation reaction.
1. Succinate is oxidised to fumarate by succinate dehydrogenase. The hydrogens are accepted by FAD to form FADH2 as the free energy change is insufficient to reduce NAD+.
2. Fumarate is hydrated to form L-malate. Fumarase catalyses a stereospecific trans addition of H+ and OH- so only L-malate is formed
3. Malate is oxidised to form oxaloacetate. NAD+ is the electron acceptor

Question 9

How is the citric acid cycle regulated?

Answer 9

The citric acid cycle is regulated by the presence of the products and reactants.
ATP lowers the affinity of enzyme for isocitrate by allosteric regulation