Tricarboxylic acid cycle

Question 1

What is the the TriCarboxylic Acid Cycle (TCA cycle) also called?

Answer 1

• The Citric Acid Cycle
• The Kreb’s Cycle

Question 2

TRUE or FALSE: The TCA cycle only has catabolic functions

Answer 2

FALSE

The TCA Cycle has both catabolic (degradation) and anabolic (biosynthetic) functions, i.e. it is amphibolic.

Question 3

Describe how the TCA Cycle has a central role in metabolism

Answer 3

• Degradation products of carbohydrates, lipids and amino acids are fed into the TCA cycle for oxidative metabolism to release energy.
• Metabolites from the TCA cycle can also be used to biosynthesize fatty acids, lipids and amino acids, and glucose.

Question 4

The TCA cycle is An “oxidative” process so where does it happen?

Answer 4

In the mitochondrial matrix

Question 5

How to remember what oxidation and reduction involve: Oxidation

Answer 5

Gain of oxygen

Loss of hydrogen

Loss of electrons

Oxidation Is Loss-OIL

Question 6

How to remember what oxidation and reduction involve: Reduction

Answer 6

Loss of oxygen

Gain of hydrogen

Gain of electrons

Reduction Is Gain of electrons-RIG

Question 7

NAD+ and FAD as key Redox Co-factors

Answer 7

NAD+/NADH and FAD / FADH2 can also be considered as “co-substrates”.

Question 8

The TCA Cycle as a central hub

Answer 8

Question 9

How does glycolysis connect to the TCA cycle?

Answer 9

• Pyruvate is transported into the mitochondrial matrix by a membrane carrier called the pyruvate translocase.
• In the matrix the next step, catalysed by pyruvate dehydrogenase (PDH) occurs.

Question 10

The Link from Glycolysis to the TCA Cycle is Pyruvate Dehydrogenase

Answer 10

• This is a REDOX reaction, called an oxidative decarboxylation.
• The acetate unit within pyruvate is activated by linking it to Coenzyme A, so that it can then undergo further reactions.

Question 11

How is Pyruvate Dehydrogenase (PDH) a Multi-Subunit Complex?

Answer 11

E₁ – Pyruvate dehydrogenase (decarboxylase)

E₂ – Dihydrolipoamide acetyltransferase

E₃ – Dihydrolipoamide dehydrogenase

Question 12

PDH requires several cofactors:

Answer 12

• Thiamine pyrophosphate (reacts with pyruvate to help cleave –CO₂)
• Lipoic Acid (passes acetate to CoA)
• Coenzyme A (accepts the acetate)
• FAD (regenerates/oxidizes lipoic acid)
• NAD+ (regenerates/oxidizes FAD)

Question 13

How is PDH an important rate-controlling step?

Answer 13

It is controlled both allosterically and by phosphorylation

Question 14

Explain the ttructure/organization of the PDH Complex

Answer 14

• The Core of PDH consists of 24 E2 chains (2 per side of a cube to form a small sphere).
• 24 E1 subunits (also 2 per side of a cube) form a larger sphere around the E2 core.
• 12 E3 submits (2 per face of a cube) associate around the E1 sphere.

Question 15

Function of co-factors in PDH?

Answer 15

• Pyruvate donates a 2-C acetate group to TPP, which passes it to oxidized lipoic acid (-S-S- form), which then passes it to CoA to form acetyl-CoA.
• FAD oxidizes the reduced lipoic acid (2-SH form) to recycle it.
• NAD+ oxidizes the FADH2 to recycle it.
• NADH is a co-product.

Question 16

For each acetyl CoA which enters the cycle:

Answer 16

(1) Two molecules of CO₂ are released (oxidative decarboxylations).
(2) Coenzymes NAD⁺ and FAD (CoQ) are reduced
(3) 3 NADH and 1 FADH₂
(4) 1 GDP (= 1 ADP) is phosphorylated to GTP
(5) The initial acceptor molecule (oxaloacetate) is reformed.

Question 17

Draw the enzymatic steps in the TCA cycle

Answer 17

1. Oxaloacetate + Acetyl CoA(Citrate synthase)→ Citrate
2. Citrate (dehydration and hydration-aconitase)→ Isocitrate
3. Isocitrate + (Isocitrate dehydrogenase- decarboxylation)→ α-ketoglutarate +NADH+ CO₂
4. α-ketoglutarate (α-ketoglutarate dehydrogenase→ Succinyl-CoA+NADH
5. Suucinyl-CoA (succinate thiokinase)→Succinate
6. Succinate (succinate dehydrogenase)→Fumarate+FADH₂
7. Fumarate (fumarase+H₂O)→L-Malate
8. L-Malate (malate dehydrogenase)→Oxaloacetate

Question 18

TCA cycle: table of enzymes & reactions

Answer 18

Question 19

Draw the structures of the TCA cycle metabolites

Answer 19

Question 20

What is the energy output per acetyl-CoA?

Answer 20

• 3 NADH
• 1 FADH₂ (CoQH₂)
• 1 GTP

Question 21

TRUE or FALSE: The 2 carbon dioxides released do not come from the acetyl-CoA in the first cycle

Answer 21

TRUE

They are lost in subsequent cycles

Like a snake that grows from the head and eats its tail…..

Question 22

How is Pyruvate dehydogenase (PDH) regulated allosterically?

Answer 22

Inhibited by NADH & Acetyl-CoA, activated by NAD+ & CoA-SH

Inhibitated by its products and activated by its substrates

Question 23

How is Pyruvate dehydogenase is regulated by phosphorylation?

Answer 23

Pyruvate dehydogenase is regulated by phosphorylation to an inactive form, catalysed by pyruvate dehydrogenase kinase (PDK):

this is activated by NADH & acetyl-CoA, inhibited by ADP & pyruvate. Pyruvate dehydrogenase phosphatase reactivates PDH.

Question 24

What is citrate synthase inhibited by?

Answer 24

Citrate synthase is inhibited by ATP

Question 25

What is isocitrate DH activated by?

Answer 25

It is activated by ADP & Ca₂+, inhibited by NADH

Question 26

What is α-Ketoglutarate DH inhibitied by?

Answer 26

It is inhibited by NADH and Succinyl-CoA; activated by Ca₂⁺

Question 27

Explain how high concentrations of PDH products switch off the enzyme, while high substrate and low energy status switch it on?

Answer 27

The unphosphorylated form of PDH is active; the phosphorylated from is inactive. The interconversion is catalysed by PDH kinase (uses ATP to add P) and PDH phosphatase (hydrolyses P). There are several isoforms of PDK.

In mammalian cells:

PDH kinase is activated by ATP, NADH and acetyl-CoA (i.e. the products of PDH), so these induce formation of the inactive form of PDH

PDH kinase is inactivated by ADP, NAD+, CoA-SH and pyruvate (i.e. substrates), so these maintain the active form of PDH.

Question 28

What are diseases of the TCA cycle?

Answer 28

A group of rare human genetic diseases that affect basic mitochondrial metabolism:

• Fumarase deficiency
• Succinate dehydrogenase deficiency
• Alpha-ketoglutarate dehydrogenase deficiency

Question 29

Diseases of the TCA cycle are a group of rare human genetic diseases that affect basic mitochondrial metabolism: Fumarase deficiency

Answer 29

Causes developmental delay, severe mental retardation, language impairment, seizures and dysmorphic facial features.

Question 30

Diseases of the TCA cycle are a group of rare human genetic diseases that affect basic mitochondrial metabolism: Succinate dehydrogenase deficiency

Answer 30

• Affects mitochondrial complex II, which links the TCA cycle with the electron transport chain.
• The phenotype is highly variable and can include Leigh syndrome, leukodystrophy, cardiomyopathy and mental and motor skill deterioration.

Question 31

Diseases of the TCA cycle are a group of rare human genetic diseases that affect basic mitochondrial metabolism: Alpha-ketoglutarate dehydrogenase deficiency

Answer 31

Very rare and characterised by encephalopathy and hyperlactatemia resulting in death in early childhood.

Question 32

TRUE or FALSE: Dysfunction or dysregulation of TCA cycle enzymes is also linked to Alzheimer’s disease and cancer.

Answer 32

TRUE

Cancer cells have altered oxidative metabolism (Warburg effect). Decreased activity of citrate synthase may be involved in metastasis

Question 33

What is the Warburg Effect in Cancer Cells?

Answer 33

• The differences between oxidative phosphorylation, anaerobic glycolysis, and aerobic glycolysis (Warburg effect).
• In the presence of oxygen, nonproliferating (differentiated) tissues first metabolize glucose to pyruvate via glycolysis and then completely oxidize most of that pyruvate in the mitochondria to CO₂ during the process of oxidative phosphorylation.
• Because oxygen is required as the final electron acceptor to completely oxidize the glucose, oxygen is essential for this process.
• When oxygen is limiting, cells can redirect the pyruvate generated by glycolysis away from mitochondrial oxidative phosphorylation by generating lactate (anaerobic glycolysis).
• This generation of lactate during anaerobic glycolysis allows glycolysis to continue (by cycling NADH back to NAD+), but results in minimal ATP production when compared with oxidative phosphorylation. Warburg observed that cancer cells tend to convert most glucose to lactate regardless of whether oxygen is present (aerobic glycolysis). This property is shared by normal proliferative tissues. Mitochondria remain functional and some oxidative phosphorylation continues in both cancer cells and normal proliferating cells.
• Nevertheless, aerobic glycolysis is less efficient than oxidative phosphorylation for generating ATP. In proliferating cells, ~10% of the glucose is diverted into biosynthetic pathways upstream of pyruvate production

Question 34

Draw the routes leading to and from the TCA cycle

Answer 34

This is a simplification.

Many of these reactions are compartmentalized; e.g. fatty acid oxidation (catabolism) takes place in the mitochondria but fatty acid synthesis is carried out in the cytosol.

The main message is that the TCA cycle supplies substrates for other (anabolic) pathways, and all major nutrient types feed into it