CAC Flashcards
When and by who was discovered the CAC?
1937
- Albert Szent-Györgyi → respiration
- Hans Krebs → CAC
What does it mean for the CAC to be amphibolic?
It is a site of anabolism and catabolism
What part of the CAC corresponds to anabolism and catabolism?
Anabolism → CAC intermediates are the starting point of anabolic pathways (ex: gluconeogenesis, fatty acid synthesis, amino acid synthesis)
*Mostly in cancer cells, highly proliferating cells, adipose cells making fat from citrate
Catabolism → CAC intermediates are the end point of catabolic pathways
- Aerobic catabolism of carbs, lipids and aa merge into the CAC (ex: oxaloacetate ↔ AA)
*Catabolic = casse
What is a Cataplerotic reaction vs an anaplerotic reaction from the stand point of the CAC?
Cataplerotic (cata = emtpying) → depletes the CAC intermediates → decreases cycle chain
*Ex: fatty acid synthesis uses citrate
Anaplerotic (ana = filing up) → replenish the depleted CAC intemediates
*Ex: Pyruvate carboxylase makes oxaloacetate
*CAC intermdediates are simple compounds so Cataplerotic reactions are part of anabolism of the cells and Anaplerotic reactions are part of catabolism of the cell
What is the Pyruvate Carboxylase reaction?
CO2 + Pyruvate → Oxaloacetate
*Replenishes the last step to allow the cycle to run again (anaplerotic)
What are the main function of the CAC?
- Producing reducing equivalents
- Produce intermediates for biosynthesis
- Produce ATP
Does the CAC harvest energy?
Is the CAC aerobic or anaerobic?
Yes, through electric gradients
Neither!! → Just an enzymatic pathway
*Could say anaerobic
What is the overall reaction of the CAC?
3 NAD+ + FAD + GDP + Pi + acetyl-CoA → 3 NADH + FADH2 + GTP + CoA + 2CO2
Which steps confer directionality to the CAC?
*Also the 3 regulated steps
Step 1 - Citrate synthase
Step 3 - Isocitrate dehydrogenase
Step 4 - a-Ketoglutarate dehydrogenase
*∆G < 0
What happens in Step 1 of the CAC?
What is the ∆G?
Oxaloacetate + Acetyl-CoA → Citrate
*Citrate Synthase
∆G˚ = -31.5kJ/mol
Citryl-CoA contains a thioester high-energy bond → hydrolysis of this bond is ∆G˚=-31.5 kJ/mol
Makes the reaction irreversible
Low pool of Oxaloacetate which would reduce the ∆G in vivo, but used readily because of the breakage of the thioesther bond
What is the only reaction where a C-C bond is formed?
Step 1: Oxaloacetate (4C) + Acetyl-CoA (2C) → Citrate
- Enol of Acetyl-CoA attacks carbonyl C in oxaloacetate
What reaction occurs at step 2 of the CAC?
Aconitase
Citrate ↔ Isocitrate (∆G˚ ~ 0kJ/mol)
Isomerization of citrate to isocitrate with cis-aconitate as an intermediate
1) Dehydration of Citrate
2) Re-hydration of cis-aconitate
What reaction occurs at step 3 of the CAC?
Isocitrate dehydrogenase
Isocitrate + NAD+ →a-ketoglutarate + CO2 + NADH + H+ (∆G˚ = -21kJ.mol)
*This step is highly inhibited by NADH
1) Dehydrogenase activity generates NADH
2) Decarboxylase activity generates CO2
3) CO2 comes from oxaloacetate, not from acetyl-CoA (exergonic, irreversible)
What happens in step 4 of the CAC?
Ketoglutarate dehydrogenase
a-ketoglutarate + CoA + NAD+ → succinyl-CoA + CO2 _ NADH + H+ (∆G˚ = -33 kJ/mol)
- Oxidation decarboxylation generates NADH and CO2
- Decarboxylation provides the energy to generate high energy intermediate: Succinylcholine-CoA
*Just like PDH, a-KGDH has E1/E2/E3 (very similar)
What happens in step 5 of the CAC?
Succinyl-CoA Synthase
Succinyl-CoA + GDP + Pi ↔ succinate + GTP (∆G˚ ~ 0kJ/mol)
- Use of the high energy succinyl-CoA to generate ATP (exergonic + endergonic cancel out)
- Energy of succinyl-CoA is conserved through succinyl-phosphate, 3-phospho-His residue, then GTP (in some cells, make ATP directly)
→ At this point, 1 equivalent acetyl-CoA (2C) has been completely oxidized to 2x CO2
→ 2x NADH and 1 GTP have been generated
What happens in step 6 of the CAC?
Succinate Dehydrogenase
Succinate + E-FAD ↔ fumarate E-FADH2 (∆G ~ 0kJ/mol)
- SDH His residue is covalently bound to FAD (FAD can’t diffuse as free metabolite)
- Dehydrogenation oc succinate produed SDH-FADH2
- SDH = Complex II of ETC → SDH-FADH2 restores FAD by feeding e- in ETC
What happens in step 7 of the CAC ?
(Fumarase)
Fumarate + H2O ↔ malate (∆G ~ 0kJ/mol)
Fumarase catalyzes the hydration of the double bond of fumarate to generate malate
What happens in step 8 of the CAC?
Malate + NAD+ ↔ oxaloacetate + NADH + H+ (∆G˚ = +29.7kJ/mol; ∆G ~ 0kJ/mol)
Although the reaction is endergonic (∆G˚ = +29.7kJ/mol), the tue ∆G ~ 0kJ/mol because in vivo, at equilibrium [Malate]»_space;» [Oxaloacetate]
The next reaction (Citrate Synthase reaction) is highly exergonic (∆G˚ = -31.5kJ/mol), which allows formation of Citrate to be exergonic even at low [oxaloacetate]
*Coupling of Step 1 and 8 helps CAC go forward (Oxaloacetate produced in step 8 is immediatly sucked to step 1)
How many ATPs are made in aerobic glycolysis vs anaerobic glycolysis?
Anaerobic glycolysis = 2 ATP
Aerobic glycolysis = 2 ATP + 5 ATP (from 2 NADH in ETC) = 7 ATP
What is the net energy production / cycle of the CAC?
3 NADH (2.5ATP/NADH) → 7.5 ATP
1 FADH2 (1.5ATP/FADH2) → 1.5 ATP
1 GTP → 1 ATP
Total/cycle = 10 ATP
Total/glucose = 2 cycles = 20 ATPs in the CAC
What is the net energy production of PDH (PDC)?
1 NADH (x2.5ATP/NADH) = 5 ATP/Glucose
What are the different mechanisms of regulation of the CAC?
(not specific steps)
- Substrate availability
- Product inhibition → product binding to the active site
- Competitive feedback inhibition → In the active site
- Allosteric activation → in a distant site
- Allosteric inhibition → in a distant site
What is the central metabolite responsible for regulation of the CAC?
NADH
Reoxidation of NADH to NAD tightly coupled to oxygen consumption and ATP synthesis
→ CAC is regulated by feedback mechanisms that coordinate its production of NADH with energy expenditure
How does Succinyl-CoA regulate the Citrate synthase step?
Citrate synthase has Acetyl-CoA as a substrate, which is very similar to Succinyl-CoA (CoA is what takes the most place) → competitive feedback inhibition as SCoA can bind to active site
How is the Citrate Synthase step regulated by substrate availability?
Acetyl-CoA and oxaloacetate
- In vivo, [Acetyl-CoA] and [oxaloacetate] do NOT saturate
The reaction rate varies on both concentrations (availability)
*[Acetyl-CoA] is controlled by PDC
How is the Citrate Synthase step regulated by product inhibition?
Citrate = product of the reaction → competitive inhibitor of oxaloacetate binding site to citrate synthase
*If citrate builds up, it will bind to the active site and block oxaloacetate’s binding
How is the Citrate Synthase step regulated by
1. Competitive feedback?
2. Allosteric inhibition?
3. Allosteric activation?
- Competitive feedback: Succinyl-CoA competes with Acetyl-CoA site
- Allsoteric inhibition: NADH (not a product)
- Allosteric activation: ADP
*Allosteric binds to distant site on Citrate Synthase
What regulates Isocitrate Dehydrogenase?
Allosteric activation → ADP and Ca2+
*Associated with exercise
Product inhibition → NADH which displaces NAD+ (at its active)
*Because Isocitrate Dehydrogenase (step 3) produced NADH
What happens when isocitrate dehydrogenase is turned OFF?
Isocitrate accumulates and is then reconverted back to citrate as step 2 is a reversible reaction → citrate goes to the cytoplasm
Citrate in the cytoplasm:
- Activates Acetyl-CoA carboxylase → activates fatty acid synthesis
- Inhibits PFK → inhibits glycolysis
(example of the CAC being Amphibolic)
What is a surplus of citrate an indicator of?
It is an indicator of a high energy charge
How is regulation of a-ketoglutarate dehydrogenase done in the CAC?
Product inhibition → NADH, Succinyl-CoA
Allosteric activation → Ca2+
What metabolite regulates Pyruvate carboxylase reaction?
Alosteric activator → Acetyl-CoA
Pyrvate + CO2 → Oxaloacetate
*When Acetyl-CoA accumulates, it needs an oxaloacetate to enter the CAC
*Pyruvate is the most important branch point in the metabolism of a cell that live on carbohydrates!!!
Wha are examples of cataplerotic reactions in the CAC?
Cataplerotic reactions are used for anabolism (Empty the CAC)
- Glucose biosynthesis → from oxaloacetate
- Fatty acid biosynthesis → Starts with Acetyl-CoA (can’t be transported out of the mitochondria so needs to go to Citrate to be transported out → enzyme breaks citrate to release Acetyl-CoA)
- Amino acid biosynthesis → Transamination of Oxaloaxetate → Aspartate // a-Ketoglutarate → Glutamate (both reversible)
What is the major source of free energy in aerobic organisms?
The CAC
Why are anaplerotic reactions critical?
The CAC can’t be interrupted so intermediates drawn off must be replenished
What are 3 important anaplerotic reactions in the CAC?
Anaplerosis → replenishes the CAC
- Pyruvate carboxylase: Pyruvate (3C) + CO2 → Oxaloacetate (4C)
- Pyruvate dehydrogenase: Pyruvate (3C) → Acetyl-CoA (2C) + CO2
- Transaminase: Pyruvate → Alanine coupled with Glutamate → a-ketoglutarate The N from glutamate is transfered to make alanine
What is the reaction equation of Pyruvate Carboxylase?
Pyruvate + HCO3- + ATP ↔ Oxaloacetate + ADP + Pi + 2H+
*Requires ATP
In vivo, concentration of oxaloacetate is so low, it really only goes forward
What are different ways Oxaloacetate can be replenished in the CAC?
- Pyruvate carboxylase: Pyruvate + CO2
- Transamination: Aspartate ↔ Oxaloacetate
- PEP carboxylase in plants and bacteria: Phosphoenol-pyruvate ↔ Oxaloacetate
How can Malate be replenished in the CAC?
Malic enzyme: Pyruvate (3C) + HCO3- + NADPH + H+ ↔ L-Malate + NADP+ + H2O
The forward replenishes the CAC (Anaplerosis)
The reverse produces NADPH for fatty acid synthesis (Cataplerosis)
*Malate eventually goes to oxaloacetate in the CAC so replenishes Malate will replenish Oxaloacetate (an all other intermediates)
What are the main transmination pairs in the CAC?
What is a transaminase reaction?
Glutamate ↔ a-Ketoglutarate
Aspartate ↔ Oxaloacetate
(Pyruvate ↔ Alanine)
Transaminase = transfer of an amine group
*In the CAC is allows C backbones to be burned as energy when there is AA build-up
What is the importance of Glutamate dehydrogenase?
How is it regulated?
GDH can act as a transaminase
Glutamate + NAD+ + H2O → a-Ketoglutarate + NH4+ + NADH + H+
*Can go both ways, but the reverse is highly unfavourable because it releases Ammonia which binds very weakly to GDH so low chances of it binding as a substrate for the reverse reaction (would need extra high concentration)
ATP = allosterical inhibitor of GDH (high energy levels)
ADP = allosterical activator of GDH
