Citric Acid Cycle

Question 1

3 subunits of pyruvate dehydrogenase complex

Answer 1

E1 (pyruvate dehydrogenase with TPP coenzyme)
E2 (dihydrolipoamide acetyltransferase)
E3 (dihydrolipoamide dehydrogenase)

Question 2

Pyruvate dehydrogenase mechanism

Answer 2

1. Decarboxylation of pyruvate
2. Transfer of hydroxyethyl group to lipoamide (E2 subunit)
3. Acetyl transfer to CoA
4. Reoxidation of lipoamide (E3 subunit’s FAD is reduced to FADH2)
5. Reoxidation of FADH2 to FAD

Question 3

Are the 2 carbons from acetyl CoA the 2 that are lost as CO2 in the citric acid cycle?

Answer 3

No- the 2 that are lost as CO2 come from oxaloacetate

Question 4

Net reaction of the citric acid cycle

Answer 4

Acetyl CoA + 2 H2O + OH- + Pi + GDP -> 2 CO2 + HS-CoA + 7 H+ + 8 electrons (6 electrons from 3 NAD+ and 2 electrons from Q) + GTP
8 electrons -> 4 oxidations x 2 electrons/oxidation

Question 5

Step 1 of citric acid cycle

Answer 5

Entry of substrate by condensation with oxaloacetate
Oxaloacetate + acetyl CoA -> Citrate + HS-CoA + H+
Enzyme: citrate synthase

Question 6

How citrate synthase works

Answer 6

Citrate synthase has 2 domains: small and large
Oxaloacetate binding causes 20 degree rotation of small domain, creating acetyl CoA binding site (ensures that acetyl CoA isn’t hydrolyzed without purpose)

Question 7

Step 2 of citric acid cycle

Answer 7

Rearrangement
Citrate -> isocitrate
Enzyme: aconitase
Reason: tertiary alcohol can’t be oxidized, but a secondary one can

Question 8

Stereoselectivity of step 2

Answer 8

3 point attachment of substrate, citrate, to chiral enzyme, aconitase
2R, 3S-isocitrate is only isomer formed

Question 9

Step 3 of citric acid cycle

Answer 9

First oxidative decarboxylation
Isocitrate + NAD+ -> alpha-ketoglutarate + NADH + CO2
Enzyme: isocitrate dehydrogenase
Secondary alcohol is oxidized to ketone

Question 10

Step 4 of citric acid cycle

Answer 10

Alpha-ketoglutarate + HS-CoA + NAD+ -> succinyl CoA + CO2 + NADH
Enzyme: alpha-ketoglutarate dehydrogenase complex
Alpha-ketoacid is coupled to CoA with the loss of CO2 and the formation of a high-energy thioester

Question 11

Step 5 of citric acid cycle

Answer 11

Succinyl CoA + GDP (or ATP) + Pi -> Succinate + GTP (or ATP) + HS-CoA
Enzyme: succinyl-CoA synthetase
Hydrolysis of high-energy thioester is coupled to formation of GTP or ATP (substrate level phosphorylation)

Question 12

3 step mechanism of step 5

Answer 12

1. Thioester phosphorylysis
2. Phosphoryl transfer to enzymatic His residue
3. Phosphoryl transfer to GDP or ADP

Question 13

Step 6 of citric acid cycle

Answer 13

Succinate + Q -> Fumarate + QH2
Enzyme: succinate dehydrogenase complex
Oxidation of alkane to trans-alkene, loss of 2 H+ and 2 electrons

Question 14

Step 7 of citric acid cycle

Answer 14

Fumarate + H2O -> L-malate
Enzyme: Fumarase
Trans-addition of water to fumarate

Question 15

Step 8 of citric acid cycle

Answer 15

L-malate + NAD+ -> oxaloacetate + NADH + H+
Enzyme: malate dehydrogenase
Secondary alcohol is oxidized to a ketone

Question 16

ATP generated per NADH

Answer 16

2.5

Question 17

ATP generated per QH2

Answer 17

1.5

Question 18

ATP per acetyl CoA

Answer 18

10 (3 NADH x 2.5 ATP/NADH + 1 GTP or ATP + 1 QH2 x 1.5 ATP/QH2)

Question 19

ATP per glucose

Answer 19

32 (Glucose -> 2 pyruvate: 2 NADH -> 5 ATP, 2 ATP; 2 pyruvate -> 2 acetyl CoA: 2 NADH -> 5 ATP; citric acid cycle: 6 NADH -> 15 ATP, 2 QH2 -> 3 ATP, 2 ATP)

Question 20

Key points of regulation for citric acid cycle

Answer 20

1. Pyruvate dehydrogenase complex
2. Citrate synthase
3. Isocitrate dehydrogenase
4. Alpha-ketoglutarate dehydrogenase complex