Lecture 5 Reactions of the Citric Acid Cycle

Question 1

How many reactions does the citric acid cycle have?

Answer 1

8 reactions with 8 enzymes mediating the cycle

Question 2

Reaction 1 of the CAC

Answer 2

Condensation reaction

the acetyl group of acetyl-CoA condenses with oxaloacetate to form citrate, catalyzed by citrate synthase.

CoA-SH and heat are released.

Question 3

What kind of condensation reaction is reaction 1 of the CAC?

Answer 3

• A Claisen condensation reaction → one ester and a carbonyl compound are linked in a carbon-carbon bond

Question 4

Thermodynamics of CAC reaction 1

Answer 4

∆G’o = -31.5 kJ/mol.

• Though exergonic because of the hydrolysis of the thioester under standard conditions, the citrate synthase reaction operates near equilibrium in vivo (∆G’ ~ 0) because the cells keep [oxaloacetate] very low.

Question 5

Reaction 2 of the CAC

Answer 5

Citrate is isomerized by dehydration to an intermediate, cis-aconitate, and then rehydration to isocitrate, all catalyzed by aconitase.

Question 6

What is the enzyme aconitase sensitive to? why?

Answer 6

Oxygen levels → therefore REDOX reactions

• Aconitase has an iron-sulfur (FeS) center, in which 1 electron redox is possible (Fe3+ to Fe2+). => reactive oxygen species (ROS) can impair the flux through the citric acid cycle and, therefore, energy metabolism.

Question 7

Thermodynamics of reaction 2 of CAC

Answer 7

Endergonic → ∆G’ᴼ = ~5 kJ/mol.

• Though endergonic, the reactions is ‘pulled’ by the following reaction which is exergonic → aconitase and isocitrate dehydrogenase are indirectly coupled.

Question 8

Reaction 3 of the CAC

Answer 8

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to form alpha-ketoglutarate.

• NAD+ is reduced to NADH.
• CO2 is released.

Question 9

What is unique about the enzyme isocitrate dehydrogenase?

Answer 9

There are 2 isoforms of the enzyme in the mitochondria; one that uses NAD+ and the other NADP+; the NAD+-dependent enzyme is in the citric acid cycle.

Question 10

Thermodynamics of reaction 3 of CAC

Answer 10

Exergonic → ∆G’o = -21 kJ/mol.

Question 11

Reaction 4 of the CAC

Answer 11

𝛼-ketoglutarate dehydrogenase catalyzes the oxidative decarboxylation to succinyl-CoA.

• NAD+ is reduced to NADH.
• CO2 is released.

Question 12

What is unique about 𝝰-ketoglutarate dehydrogenase from reaction 4 of CAC?

Answer 12

It is a multienzyme complex similar to PDH having E1, E2 and E3 enzymes and cofactors. The difference is that their E1 enzymes bind different substrates

Question 13

Thermodynamics of reaction 4 of the CAC

Answer 13

exergonic → ∆G’º = -33 kJ/mol

• helps pull the reaction forward
• the energy is conserved in the thioester bond

Question 14

Reaction 5 of the CAC

Answer 14

Succinyl-CoA synthetase uses GDP as a substrate and couples the cleavage of the high-energy thioester linkage in succinyl- CoA to the phosphorylation of GDP to yield GTP. This is a substrate-level phosphorylation reaction.

Question 15

Thermodynamics of reaction 5 of the CAC

Answer 15

exergonic → ∆G’º = -2.1 kJ /mol .

• The reaction proceeds through a phosphorylated enzyme intermediate.

Question 16

Reaction 6 of the CAC

Answer 16

Succinate dehydrogenase, also known as succinate-ubiquinone, oxidoreductase (complex II in ETC – electron transfer chain) catalyzes the oxidation of succinate to fumarate using a covalently-attached FAD cofactor and 3 iron-sulfur clusters to reduce ubiquinone to ubiquinol.

• FAD is reduced to FADH2.

Question 17

Thermodynamics of reaction 6 of the CAC

Answer 17

endergonic → ∆G’º = +6 kJ/mol.

• Reaction is endergonic on its own, but can be pulled forward through coupling.

Question 18

What is the enzyme succinate dehydrogenase a part of?

Answer 18

integral membrane protein of the mitochondrial inner membrane

Question 19

Reaction 7 of the CAC

Answer 19

The reversible hydration of fumarate to L- malate is catalyzed by the enzyme fumarase.

Question 20

Thermodynamics of reaction 7 of the CAC

Answer 20

exergonic → • ∆G’º = -3.4 kJ/mol.

Question 21

Reaction 8 of the CAC

Answer 21

malate dehydrogenase oxidizes malate to oxaloacetate.

• NAD+ is reduced to NADH

Question 22

Thermodynamics of reaction 8 of the CAC

Answer 22

endergonic → ∆G’º = +29.7 kJ/mol.

• Under standard conditions, the reaction is highly endergonic, but in vivo the reaction is pulled as [oxaloacetate] is kept very low by the citrate synthase reaction

Question 23

What is the formation of oxaloacetate in the CAC similar to in glycolysis?

Answer 23

Similar to formation of 1,3BPG because both reaction are unfavourable to make but they are pulled forward by having there concentrations low due to following reactions that are highly favourable.

Question 24

Products of the CAC per pyruvate and glucose molecule

Answer 24

Yield per pyruvate molecule →

• 2 CO2 → reactions 3 & 4
• 3 NADH → reactions 3, 4, & 8
• 1 FADH2 → reaction 6
• 1 GTP (= 1ATP). → reaction 5

Yield per glucose molecule →

• 4 CO2 → reactions 3 & 4
• 6 NADH → reactions 3, 4, & 8
• 2 FADH2 → reaction 6
• 2 GTP (= 1ATP). → reaction 5

Question 25

Where does the CO2 in the CAC originate from?

Answer 25

The oxaloacetate, NOT the acetylCo-A.

• The way in which oxaloacetate and acetylCoA condense together, the CO2 comes off of the oxaloacete so after 2 cycles the CO2 will come from the acetylCoA as its carbons move down the oxaloacetate molecule

Question 26

How much ATP is synthesized from NADH and FADH₂ once they traverse the ECT?

Answer 26

• ~2.5 ATP/ NADH
• ~1.5 ATP/ FADH₂

Question 27

Energetics of 1 molecule of glucose through oxidation?

Answer 27

One molecule of glucose powers two turns of the CAC and yields ~32 ATP.

Question 28

What kind of pathway is the citric acid cycle?

Answer 28

Amphibolic → this cycle is connected to both oxidative (catabolic) and reductive (anabolic/biosynthetic) processes.

Question 29

How is the CAC catabolic?

Answer 29

CAC oxidizes acetyl-CoA (oxaloacetate) to CO2

Question 30

How is the CAC anabolic?

Answer 30

CAC provides precursors for:

• Gluconeogenesis: pyruvate is converted to oxaloacetate (by pyruvate carboxylase) to PEP (by PEPCK) to glucose).
• Glyceroneogenesis: as above oxaloacetate goes to PEP which enters a ‘short’ gluconeogenesis pathway to DHAP and to glycerol-3-phosphate – used for synthesis of triglycerides and glycerophospholipids.
• Lipid biosynthesis: any excess of mitochondrial acetyl-CoA is converted to citrate (citrate shuttle) that leaves the cycle to be reconverted to acetyl-CoA (in the cytosol), which is the basis for the synthesis of fatty acids and sterols.

Question 31

What sustains the CAC to keep going?

Answer 31

Balance between cataplerosis and anaplerosis

• Anaplerotic reactions replenish TCA cycle intermediates to ensure its continued function.
• Cataplerotic reactions dispose of TCA cycle intermediates that are in excess
• If intermediates are added to the TCA cycle, their removal avoids their excessive accumulation.

Question 32

What vitamins play a role in the CAC?

Answer 32

Vitamins such as riboflavin, niacin, and thiamine work as coenzymes in this cycle, while pantothenic acid forms the CoA part of acetyl-CoA.

Question 33

Can animals make glucose from fat?

Answer 33

No because although fat can be biosynthesized to Acetyl-CoA, you cannot make oxaloacetate directly from Acetyl-CoA and fat cannot be biosynthesized to oxaloacetate.

• plants and microorganisms can however via the glyoxylate cycle

Question 34

What 3 factors regulate the CAC?

Answer 34

• substrate availability → OAA and acetyl-CoA.
• product inhibition → IDH and 𝛼- KDH are strongly inhibited by NADH; citrate synthase is inhibited by citrate.
• feedback inhibition → succinyl- CoA inhibits citrate synthase.

Question 35

What three CAC enzymes carry out highly favourable reactions?

Answer 35

Three CAC enzymes operate far from equilibrium (irreversible reactions):

• citrate synthase (CS)
• isocitrate dehydrogenase (IDH)
• 𝛼-ketoglutarate dehydrogenase (𝛼-KDH).