Lecture 4 (W2) - TCA cycle

Question 1

What do dehydrogenases (enzyme) do?

Answer 1

Dehydrogenases catalyse the oxidation of a substrate through the transfer of hydrogen from a substrate to an electron acceptor such as NAD⁺ or FAD

Question 2

Before entering the TCA cycle, what is pyruvate converted into? What enzyme is involved and is there any electron acceptor involved?

Answer 2

Pyruvate (3C) → Acetyl-CoA (2C)

• Catalysed by pyruvate degydrogenase
• NAD⁺ is converted into NADH + H⁺

**Note : whenever there is decarboxylation (removal of C atom) in TCA, it often means the substrate is oxidised, so there must be electron carrier such as NAD⁺ or FAD to be reduced. This is why its called OXIDATIVE DECARBOXYLATION **

Question 3

What is the first irreversible step in the TCA cycle? Also list enzyme involved and electron acceptor (if any)

Answer 3

Acetyl CoA (2C) + oxaloacetate (4C) → citrate
- Catalysed by citrate synthase
- No electron acceptor involved

Question 4

The second irreversible reaction, isocitrate (6C) → α-ketoglutarate (5C) + CO₂ is catalysed by which enzyme? Any electron carriers involved?

Answer 4

• Catalysed by isocitrate dehydrogenase
• NAD⁺ is converted into NADH + H⁺

Question 5

The third irreversible reaction, α ketoglutarate (5C) + CoASH → succinyl-CoA (4C) + CO₂, is catalysed by which enzyme? Any electron carriers involved?

Answer 5

• Catalysed by α ketoglutarate dehydrgenase.
• Involves conversion of NAD⁺ into NADH + H⁺

Question 6

Which other reaction in the TCA cycle involves NAD⁺? List it out

Answer 6

l-malate (alcohol) → oxaloacetate (ketone)
- catalysed by malate dehydrogenase

Note : no release of CO₂

Question 7

Which reaction in the TCA cycle involves FAD as an electron carrier? List the reaction out and state the enzyme involved

Answer 7

Succinate (C-C) → Fumarate (C=C)
- catalysed by succinate dehydrogenase

Note : no release of CO₂

Question 8

How are the 3 irreversible reactions in the TCA cycle regulated?

Answer 8

Through allosteric regulation

Question 9

Irreversible reaction 1 : Acetyl CoA (2C) + oxaloacetate (4C) → citrate.

What are the activators and inhibitors of this reaction?

Answer 9

Activators : -
Inhibitors : ATP, NADH, citrate (product inhibition)

Question 10

Irreversible reaction 2 : isocitrate (6C) → α-ketoglutarate (5C) + CO₂

What are the activators and inhibitors of this reaction?

Answer 10

Activators : Ca²⁺, ADP
Inhibitors : ATP

Question 11

Irreversible reaction 3 : α ketoglutarate (5C) + CoASH → succinyl-CoA (4C) + CO₂

What are the activators[1] and inhibitors [2] of this reaction?

Answer 11

Activators : Ca^{2+
Inhibitors: succinyl-CoA (product inhibition), NADH}

Question 12

The second form of regulation in the TCA cycle is substrate availability. Explain how this works.

Oxaloacetate (OAA)

Answer 12

The rate of TCA cycle is determined by availability of substrates, esp oxaloacetate (OAA).
- OAA can be transported out of the mitochondria by being converted into malate / aspartate and then going through the malate / aspartate shuttle. OAA is regenerated in the cytosol to be utilised in gluconeogenesis.
- Thus, if OAA is transported out of the mitochondria, it limits its availability and slows down rate of TCA cycle in the mitochondria.

Question 13

The third layer of regulation of the TCA cycle is enzyme complexes. Explain how enzyme complexes act as a form of regulation.

Answer 13

Enzyme complexes allow for improved efficiency and coordinated control of metabolic pathways as enzymes are physically grouped together instead of working independently.

Question 14

How do enzyme complexes improve efficiency?

Answer 14

They significantly reduce the diffusion distance of substrates from one enzyme to another, allowing faster reactions. (improved efficiency as substrates do not need to diffuse long distances, as compared to when enzymes are independent of each other)

Question 15

How do enzyme complexes prevent side reactions?

Answer 15

Enzyme complexes keep intermediates within the complex, preventing unwanted side reactions from occuring.

Question 16

Which enzyme is a enzyme complex and what reaction does it catalyse?

1. Isocitrate dehydrogenase
2. Pyruvate dehydrogenase
3. α ketoglutarate dehydrogenase
4. Malate dehydrogenase

Answer 16

2. Pyruvate dehydrogenase
   - catalyses the reaction : pyruvate (3C) → acetyl-CoA (2C) + CO₂

Question 17

How many enzyme complexes is pyruvate dehydrogenase made up of? How many coenzymes does it have?

Answer 17

• 3 enzyme complexes : E1, E2 and E3
• 5 different coenzymes

Question 18

What are the 3 modes of regulation of pyruvate dehydrogenase?

Answer 18

1. Formation of enzyme complex (PDC, pyruvate dehydrogenase complex)
2. Allosteric inhibition by products, such as NADH and acetyl-CoA
3. Covalent modification (phosphorylation of enzyme complex E1)

Question 19

What enzymes are involved in phosphorylation / dephosphorylation of enzyme complex E1, pyruvate dehydrogenase?

Answer 19

1. E1 phosphatase
2. E1 kinase : adds phosphate group to pyruvate dehydrogenase, preventing substrate binding.

Question 21

In the TCA cycle (1 glucose molecule), how many NADH / FADH₂ / ATP is produced? Include the pyruvate → acetyl CoA step

Answer 21

For each pyruvate:
- pyruvate → acetyl CoA = 1 NADH
- 3 NADH, 1 FADH₂, 1 GTP (~ATP)

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Thus, for each glucose :
- 4 x 2 = 8 NADH
- 2 FADH₂
- 2 GTP

Question 22

Given that :
- 1 NADH = 3ATP
- 1 FADH₂ = 2 ATP

How many ATP molecules are generated from both glycolysis and TCA cycle for 1 glucose molecule?

Answer 22

**Glycolysis : net 2 ATP + 2 NADH (6 ATP) = 8 ATP **
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TCA cycle
- 8 NADH = 24 ATP
- 2 FADH₂ = 4 ATP
- 2 GTP = 2 ATP
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Total = 38 ATP