M2: Citric Acid Cycle L10

Question 1

What are alternate names for the CAC?

Answer 1

TCA cycle or Krebb’s cycle.

Question 2

What does it mean to be amphibolic?

Answer 2

Amphibolic = site of anabolism and catabolism

Question 3

What is anabolism in the CAC? Give examples.

Answer 3

CAC intermediates are the starting point of anabolic pathways.
Ex: gluconeogenesis, fatty acid synthesis (to make membranes), and amino acid synthesis (to make proteins).

Question 4

What type of reaction are the anabolic processes that stem from the CAC? Why?

Answer 4

The anabolic reactions are Cataplerotic reactions (cata = emptying) because they deplete the CAC intermediates.

Question 5

What is catabolism in the CAC? Give examples.

Answer 5

CAC intermediates are the end point of catabolic pathways. The aerobic catabolism of carbohydrates, lipids and amino acids merge into the CAC.
Ex: oxaloacetate (from pyruvate carboxylase) and amino acid degradation.

Question 6

What type of reactions are the catabolic processes involving the CAC? Why?

Answer 6

The catabolic rxns are Anaplerotic reactions (ana = filling up) because they replenish the depleted CAC intermediates.

Question 7

What are the 3 main functions of the CAC?

Answer 7

1. To produce reducing equivalents (3NADH and 1FADH2): electron carriers that donate their electrons to ETC.
2. Produces intermediates for biosynthesis (citrate, fatty acids, amino acids).
3. Removes carbons to form CO2.

Question 8

Is the CAC anaerobic or aerobic?

Answer 8

Neither! It supports aerobic metabolism but doesn’t consume oxygen directly.

Question 9

Does the CAC generate energy?

Answer 9

Yes, its harvested through the reducing equivalents and It makes one molecule of GTP (which is an ATP equivalent).

Question 10

What 3 irreversible steps confer directionality to the CAC? Why?

Answer 10

Step 1: citrate synthase
Step 3: Isocitrate dehydrogenase
Step 4: Alpha-ketoglutarate dehydrogenase

Because they all have negative ∆G.

Question 11

What are the high energy steps of the CAC? What are the recycling steps of the CAC?

Answer 11

High energy: first 5 steps.

Recycling: last 3 steps.

Question 12

What is step 1 of the CAC? What is unique about this step?

Answer 12

Oxaloacetate (4 carbons) + acetyl-CoA (2 carbons) to Citrate (6 carbons) by citrate synthase.

Unique because it’s the only reaction where a C-C bond is formed.

Question 13

How is Oxaloacetate turned into citrate? Why is this step (step 1) irreversible?

Answer 13

1) Enol of acetyl-CoA attacks carbonyl C in oxaloacetate
2) Citryl-CoA is an intermediate which contains a thioester (i.e. high energy bond)

3) Hydrolysis of Citryl-CoA is associated negative free E value (∆G˚’ = - 31.5 kJ/mol)
Driving force for the reaction. Irreversible.

Question 14

Memorize the CAC.

Answer 14

Lecture 10.

Question 15

What is the overall rxn of step 2 of the CAC? What are key points of the reaction?

Answer 15

Citrate to isocitrate reversibly. Enzyme = Aconitase.
Isomerization of citrate to isocitrate, with cis-aconitate as an intermediate.
1) Dehydration of Citrate
2) Re-hydration of cis-aconitate to make isocitrate (new isomer).

Question 16

What is the overall rxn of step 3 of the CAC? What are key points of the reaction? Irreversible or reversible?

Answer 16

Step 3 is a decarboxylation via the enzyme isocitrate dehydrogenase.
Isocitrate (6 carbons) + NAD+ to a-ketoglutarate (5 carbons) + CO2 (1 carbon) + NADH + H+

Key points:
1) Dehydrogenase activity generates NADH (first time),
decarboxylase activity generates CO2 (first time).
2) CO2 comes from oxaloacetate, not from acetyl-CoA (exergonic, irreversible).
3) Rxn is irreversible.

Question 17

What is the overall rxn of step 4 of the CAC? What are key points of the reaction? Reversible or irreversible?

Answer 17

Step 4 is a decraboxylation reaction that occurs via the enzyme a-Ketoglutarate Dehydrogenase and is irreversible.

a-ketoglutarate (5 carbons) + CoA + NAD+ to succinyl-CoA (4 carbons)+ CO2 (1 carbon) + NADH + H+.

Key points:
1) Oxidative decarboxylation that generates NADH
(second time) and CO2 (second time).
2) The decarboxylation reaction provides the energy to generate the high energy intermeidate: succinyl-CoA
3) Like Pyruvate dehydrogenase, alpha-KG
dehydrogenase is a multienzyme complex.

Question 18

What are the enzymes in the ketoglutarate dehydrogenase complex?

Answer 18

E1 = ketoglutarate dehydrogenase; 
E2 = dihydrolipoyl transsuccinylase; 
E3 = dihydrolipoyl dehydrogenase

Question 19

What is the overall rxn of step 5 of the CAC? What are key points of the reaction? Reversible or irreversible?

Answer 19

Step 5 is a reversible rxn that occurs via Succinyl-CoA Synthetase.

Succinyl-CoA + GDP + Pi to succinate + GTP

Key points:

1) Use of the high energy succinyl-CoA to generate GTP.
2) Energy of succinyl-CoA is conserved through succinyl-phosphate, 3-phospho-Histidine residue, then GTP.
3) At this point, 1 equivalent acetyl-CoA has been completely oxidized to 2 CO2
4) 2 NADH, and 1 GTP have been generated

Question 20

What is the overall rxn of step 6 of the CAC? What are key points of the reaction? Reversible or irreversible?

Answer 20

Step 6 is a reversible oxidation reaction via the succinate dehydrogenase enzyme.

Succinate + E-FAD to fumarate + E-FADH2

Key points:

1) Succinate dehydrogenase is covalently bound to FAD therefore FAD cannot diffuse as a free metabolite.
2) Dehyrogenation of succinate produced Succinate dehydrogenase-FADH2
3) Succinate dehydrogenase-FADH2 restores its FAD by directly funneling electrons into ETC through complex 2.

Question 21

What step of the CAC is directly involved in the ETC?

Answer 21

Step 6: succinate to fumarate via succinate dehydrogenase. Generates FADH2 that enters ETC through complex 2.

Question 22

What is the overall rxn of step 7 of the CAC? What are key points of the reaction? Reversible or irreversible?

Answer 22

Step 7 is a reversible hydration reaction via the fumarase enzyme.

Fumarate + H2O to malate

Key point:
- Fumarase catalyzes the hydration of the double bond of fumarate to generate malate.

Question 23

What is the overall rxn of step 8 of the CAC? What are key points of the reaction? Reversible or irreversible?

Answer 23

Step 8 is a reversible reaxtion that occurs via the malate dehydrogenase enzyme.

Malate + NAD+ to oxaloacetate + NADH + H+

Key points:
1) Although the reaction is endergonic (∆G˚’ = + 29.7 kJ/mol), the true ∆G ~ 0 kJ/mol because:
In vivo: at equilibrium [Malate]&raquo_space;» [Oxaloacetate] so it pushes rxn towards oxaloacetate.

2) The next reaction (Citrate Synthase reaction) is highly exergonic (∆G˚’ = -31.5 kJ/mol), which allows the formation of citrate to be exergonic even at low [oxaloacetate]
3) Coupling of both reactions helps the CAC run forward.

Question 24

How many ATP’s are made from one round of the CAC?

Answer 24

10 ATP per acetyl-coA.
each glucose makes 2 pyruvates, which makes 2 acetyl-CoA, 
From each acetyl CoA:
3 NADH (2.5 ATP/NADH) = 7.5 ATP
1 FADH2 (1.5 ATP/FADH2) = 1.5 ATP
1 GTP (1ATP/GTP) = 1ATP
Total: 10 ATP/ acetyl CoA
which therefore generates a total of 20 ATP for 2 rounds of CAC.

Question 25

What are the 5 mechanisms for CAC regulation?

Answer 25

1- substrate availability
2- product inhibition
3- competitive feedback inhibition
4- allosteric activation
5- allosteric inhibition

Question 26

What compound inhibits all 3 key enzymes of the CAC?

Answer 26

NADH

Question 27

How is citrate synthase regulated? Explain each method.

Answer 27

Substrate availability:
- Acetyl-CoA and oxaloacetate: as their concentrations increase, it drives the reaction towards citrate production.
-In vivo, [acetyl-CoA] and [oxaloacetate]
do not saturate citrate synthase.
=> The reaction rate varies with the
concentrations of the two precursors.
[acetyl-CoA] is controlled by PDC.

Product inhibition: 
Citrate = the product of the reaction 
            = competitive inhibitor of 
               oxaloacetate binding to 
               citrate synthase

Competitive feedback:
Succinyl-CoA competes with acetyl-CoA for the citrate synthase binding site.

Allosteric inhibition:
NADH (not a product)

Allosteric activation:
ADP (ex: high exercise)

Question 28

How is isocitrate dehydrogenase regulated? Explain each method. What happens when isocitrate dehydrogenase is off?

Answer 28

Product inhibition:
NADH which displaces NAD+

Allosteric activation:
ADP and Ca2+ (associated with exercise or muscle contraction)

When isocitrate dehydrogenase is OFF:
=> isocitrate to citrate is reversible. Citrate builds up so citrate goes to cytoplasm.

Citrate in cytoplasm:

 - Activates acetyl-coA carboxylase which Activates fatty acid synthesis
 - Inhibits PFK which Inhibits glycolysis because it is inhibiting the committing step.

Question 29

What does a surplus of citrate in the cytoplasm indicate?

Answer 29

A high energy charge.

Question 30

How is a-ketoglutarate dehydrogenase regulated? Explain each method.

Answer 30

Product inhibition:
- inhibited by NADH, succinyl-CoA

Allosteric activation:
- Ca2+

Question 31

What is indicated by an accumulation of acetyl-CoA for the CAC? What will occur in response to the accumulation?

Answer 31

An accumulation of acetyl-CoA indicates that there is a need for oxaloacetate in order to restart the CAC. this will activate pyruvate carboxylase to turn pyruvate into oxaloacetate (anabolic) instead of the catabolic reaction from pyruvate to acetyl-CoA. The presence of new oxaloacetate will allow the CAC to start.

Question 32

What 3 enzymes OUTSIDE of the CAC are regulated by intermediates of the CAC to then regulate the CAC? Name the intermediates that regulate each of them.

Answer 32

1) Phosphofructokinase (inhibition by citrate)
2) Pyruvate dehydrogenase (inhibition by acetyl-CoA and NADH)
3) Pyruvate carboxylase (activation by acetyl-CoA)

Question 33

Why does NADH have a central role in regulating the CAC?

Answer 33

Because it is related to controlling the energy charge of the cell. This is because reoxidation of NADH to NAD+ is tightly coupled to oxygen consumption and ATP synthesis.
If the cell is in need of ATP, the CAC will have the greatest flux.
If the cell is in abundance of ATP and NADH, intermediates will be used for other purposes.