Lecture 4 (W2) - TCA cycle Flashcards
What do dehydrogenases (enzyme) do?
Dehydrogenases catalyse the oxidation of a substrate through the transfer of hydrogen from a substrate to an electron acceptor such as NAD+ or FAD
Before entering the TCA cycle, what is pyruvate converted into? What enzyme is involved and is there any electron acceptor involved?
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 **
What is the first irreversible step / first commited step in the TCA cycle? Also list enzyme involved and electron acceptor (if any)
Acetyl CoA (2C) + oxaloacetate (4C) → citrate
- Catalysed by citrate synthase
- No electron acceptor involved
The second irreversible reaction, isocitrate (6C) → α-ketoglutarate (5C) + CO2 is catalysed by which enzyme? Any electron carriers involved?
- Catalysed by isocitrate dehydrogenase
- NAD+ is converted into NADH + H+
The third irreversible reaction, α ketoglutarate (5C) + CoASH → succinyl-CoA (4C) + CO2, is catalysed by which enzyme? Any electron carriers involved?
- Catalysed by α ketoglutarate dehydrgenase.
- Involves conversion of NAD+ into NADH + H+
What are the 4 key enzymes in the TCA cycle that catalyses irreversible reactions and activity is regulated?
- Pyruvate dehydrogenase (pyruvate → acetyl CoA)
- Citrate synthase (oxaloacetate + acetyl CoA → citrate)
- Isocitrate dehydrogenase (Isocitrate → α-ketoglutarate)
- α-ketoglutarate dehydrogenase (α-ketoglutarate → succinyl-CoA)
Which reaction in the TCA cycle releases GTP?
Succinyl-CoA (4C) → succinate
Since the -CoA group is cleaved off, it releases energy and the reaction is exergonic. Thus, it is paired with the endergonic reaction : GDP + P → GTP
Which reaction in the TCA cycle involves FAD as an electron carrier? List the reaction out and state the enzyme involved
Succinate (C-C) → Fumarate (C=C)
- catalysed by succinate dehydrogenase
Note : no release of CO2
How are the 3 irreversible reactions in the TCA cycle regulated?
Through allosteric regulation
(& product inhibition)
Irreversible reaction 1 : Acetyl CoA (2C) + oxaloacetate (4C) → citrate.
What are the activators and inhibitors [3] of this reaction?
Activators : -
Inhibitors :
1. ATP
2. NADH (allosteric inhibitor, not product inhibitor as NADH is not produced in this step)
3. Citrate (product inhibition)
Irreversible reaction 2 : isocitrate (6C) → α-ketoglutarate (5C) + CO2
What are the activators [2] and inhibitors [1] of this reaction?
Activators : Ca2+, ADP
Inhibitors : ATP
Irreversible reaction 3 : α ketoglutarate (5C) + CoASH → succinyl-CoA (4C) + CO2
What are the activators[1] and inhibitors [2] of this reaction?
Activators : Ca2+
Inhibitors: succinyl-CoA (product inhibition), NADH
The second form of regulation in the TCA cycle is substrate availability. Explain how this works.
Oxaloacetate (OAA)
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.
The third layer of regulation of the TCA cycle is enzyme complexes. Explain how enzyme complexes act as a form of regulation.
Enzyme complexes allow for improved efficiency and coordinated control of metabolic pathways as enzymes are physically grouped together instead of working independently.
How do enzyme complexes improve efficiency?
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). This also prevents intermediates from being lost through side reactions.
How do enzyme complexes prevent side reactions?
Enzyme complexes keep intermediates within the complex, preventing unwanted side reactions from occuring.
Which enzyme(s) are multi-enzyme complex and what reaction does it catalyse?
- Isocitrate dehydrogenase
- Pyruvate dehydrogenase
- α-ketoglutarate dehydrogenase
- Malate dehydrogenase
- Pyruvate dehydrogenase
- catalyses the reaction : pyruvate (3C) → acetyl-CoA (2C) + CO2
AND - α-ketoglutarate dehydrogenase : α-ketoglutarate → succinyl-CoA
How many enzyme complexes is pyruvate dehydrogenase made up of? How many coenzymes does it have?
- 3 enzyme complexes : E1, E2 and E3
- 5 different coenzymes
What are the 3 modes of regulation of pyruvate dehydrogenase?
- Formation of enzyme complex (PDC, pyruvate dehydrogenase complex)
- Allosteric inhibition by products, such as NADH and acetyl-CoA
- Covalent modification (phosphorylation of enzyme complex E1 ; catalysed by E1 kinase/phosphatase. E1 phosphatase activates the E1 enzyme complex of pyruvate dehydrogenase)
What enzymes are involved in phosphorylation / dephosphorylation of enzyme complex E1, pyruvate dehydrogenase?
- E1 phosphatase : removes phosphate group from pyruvate dehydrogenase.
- E1 kinase : adds phosphate group to pyruvate dehydrogenase, preventing substrate binding.
How does the [NADH]/[NAD+] ratio regulate the TCA cycle?
- When [NADH]/[NAD+] ratio is high, it indicates that sufficient ATP is being generated and NADH accumulation is occuring. Thus, the rate of TCA cycle slows down as NADH inhibits key enzymes citrate synthase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase)
- Conversely, when [NADH]/[NAD+] ratio is low, TCA cycle speeds up.
In the TCA cycle (1 glucose molecule), how many NADH / FADH2 / ATP is produced? Include the pyruvate → acetyl CoA step
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
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?
**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
