MCBG Session 6 - Catabolic Pathways (Part 3)

Question 1

Summarise the 4 main steps/enzymes that are regulated in glycolysis and how they are regulated.

Answer 1

1) Step 1 - Hexokinase
- Allosteric inhibition by glucose-6-phosphate (G6P).
2) Step 3 - Phosphofructokinase (key regulator)

• High ATP (high energy signal) inhibits PFK
• High ADP (low energy signal) stimulates PFK
• High insulin stimulates PFK (assisting removal of glucose)
• High glucagon inhibits PFK (assisting increase of glucose)

3) Step 6 -
- High NADH/low NAD+ (low energy signals) inhibits step 6.
4) Step 10 - Pyruvate kinase
- High insulin:glucagon ratio stimulates pyruvate kinase via enzyme dephosphorylation.

Question 2

What is the role of pyruvate dehydrogenase in glucose metabolism?
What regulates pyruvate dehydrogenase (PDH)?

Answer 2

Pyruvate dehydrogenase converts pyruvate into AcetylCoA (via combination with CoA + NAD+) ready for the TCA (Krebs) cycle

Stimulated by low energy signals via dephosphorylation:

• Pyruvate
• Low energy signals (NAD, ADP)
• Insulin (via dephosphorylation)

Inhibited by high energy signals via phosphorylation:

• Acetyl CoA
• NADH
• ATP
• Phosphorylation

Question 3

What occurs if someone has PDH deficiency?

Answer 3

Lactic acidosis - as pryuvate builds up but doesn’t go through TCA cycle, instead is diverted and acted on by LDH to form lactate.

Question 4

• What happens in the TCA cycle?

- What is produced?

Answer 4

• Acetyl converted to 2xCO2
• Oxidative reactions, so requires NAD+/FAD
• Some energy produced
• Precursors for biosynthesis produced

Question 5

What are the key principles of the TCA cycle?

Answer 5

• 2C molecule introduced into cycle and broken down to release CO2
• Reactions oxidative so NADH/FADH2 produced
• Small amount of energy released in the form of GTP

Total production for 2 cycles (1 glucose)

• 6 x NADH (3 for each cycle)
• 2 x FADH2 (1 for each cycle)
• 2 x GTP (1 for each cycle)

Question 6

How is the TCA cycle regulated?

Answer 6

Via high + low energy signal molecules:

• ADP/NAD+ (low energy signal) stimulates
• ATP/NADH (high energy signal) inhibits

Question 7

Where does the TCA cycle occur?

What does the TCA cycle not function without?

Answer 7

• In the mitochondria

- Oxygen

Question 8

What does the TCA cycle produce precursors for?

Answer 8

For biosynthesis of other molecules, e.g.: citrate can feed out of the cycle for biosynthesis of fatty acids, oxaloacetate for amino acids and glucose etc etc

Question 9

What are the 2 processes of the final (stage 4) of glucose catabolism?

Answer 9

1) Electron transport - electrons on NADH/FADH2 transported down carrier molecules to oxygen, releasing energy in steps
2) Free energy released used to drive ATP synthesis (known as oxidative phosphorylation)

Question 10

What is the energy from movement of electrons down transport chain used for?

Answer 10

The movement of protons, driving them from the matrix to intermembrane space. This creates a proton motive force (pmf) back towards the mitochondrial matrix.

Question 11

Which enzyme (along with the proton motive force) drives the synthesis of ATP during this stage?

Answer 11

ATP synthase - protons return back across the membrane via ATP synthase which the energy for ATP synthesis.

Question 12

Oxidation of 2 moles NADH produces 5 moles ATP, but 2 moles FADH2 produces 3 moles ATP - why is this?

Answer 12

Electrons in NADH have more energy, and NADH uses 3 proteins translocating complexes (PTC’s) compared to FADH’s 2. The greater the pmf, the more ATP synthesised.

• 2 mol NADH = 5 mol ATP (2.5 mol per NADH)
• 2 mol FAD2H = 3 mol ATP (1.5 mol per FAD2H)

Question 13

How is oxidative phosphorylation regulated?

Answer 13

High ATP = inhibits - as there is no substrate (ADP) for ATP synthase, so H+ concentration in inter-membrane space increases
High ADP = stimulates

Question 14

How does cyanide (CN-)/carbon monoxide (CO) inhibit oxidative phosphorylation?

Answer 14

Blocks (inhibits) electron transport, prevents acceptance of electrons by O2, no proton motive force to drive synthesis of ATP.

Question 15

How do certain substances such as dinitrophenol uncouple oxidative phosphorylation?
What tissue in newborn infants + hibernating animals uses this uncoupling process to generate heat?

Answer 15

• Increases permeability of inner mitochondrial membrane
• Dissipates proton gradient, therefore no proton motive force for ATP synthesis - energy therefore lost as heat.
• Brown adipose tissue, uses thermogenin (UCP1), which transports H+ back into mitochondria so electron transport is uncoupled from ATP synthesis - energy from pmf released as extra heat.

Question 16

Outline the differences between oxidative and substrate level phosphorylation

Answer 16

• Oxidative PP requires membrane-associated complexes, substrate PP requires soluble enzymes
• Oxidative PP cannot occur in absence of O2, but substrate PP can occur to a limited extent
• Oxidative PP is major process for ATP synthesis in cells requiring large amounts of energy, but substrate level PP is only a minor process for these cells.