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BIOC 212 > Citric acid cycle > Flashcards

Citric acid cycle Flashcards

1
Q

What reaction si responsible for transition from pyruvate to Acetyl-CoA?

A

Pyruvate = product of glycolysis, enters the mitochondria
Acetyl-CoA = enters CAC

Happens in the mitochondria:
Pyruvate + CoA + NAD+ → (PDC) → Acetyl-CoA + CO2 + NADH + H+
PDC = pyruvate dehydrogenase complex

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2
Q

How does pyruvate enter the mitochondrion?

A

Pyruvate gets into the mitochondrion through the pyruvate translocase (H+ symport)

Symport of H+ is required for maintenance of the electrochemical gradient (pyruvate is negative)

*Also ATP/ADP antiport in mitochondrial membrane

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3
Q

What is the structure/composition of the Pyruvate Dehydrogenase Complex (PDC)?

A

PDC = Massive mutli-enzyme complex
Size = 9.5 MegaDaltons in eukaryotes (substrate = pyruvate = 88 Daltons)

Composition: (*remember)
- *E1 pyruvate dehydrogenase → 30 heterotetramers/complex
- E2 dihydrolipoyl transacetylase → 60 core monomers/complex
- E3 dihydrolipoyl dehydrogenase → 12 homodimers
- E3-binding protein
- Pyruvate dehydrogenase kinase → 1-3/complex
- Pyruvate dehydrogenase phosphatase → 1-3/complex
- *5 different coenzymes required for catalytic activities

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4
Q

What occurs in the 1st step of Pyruvate Dehydrogenase Reaction?

A

Mediated by E1 (pyruvate dehydrogenase), decraboxylation step:

Pyruvate + TPP → Hydroxyethyl-TPP + CO2 (from pyruvate, then diffused out of the mitochondria)

*Pyruvate put onto TPP as a committment
Step 1 is irreversible → whole Pyruvate dehydrogenase reaction irreversible (all other steps are reversible)

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5
Q

How does step 5 of the pyruvate dehydrogenase reaction occur?

A

E3 mediated oxidation of FAD by NADH:
FADH2+ + NAD → FAD + NADH + H+

*In FADH2, H+ are on the cysteines → FAD+ has disulfide bond

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6
Q

Which step of the pyruvate dehydrogenase reaction is responsible for synthesis of the final acetyl-CoA product?

A

3rd step (out of 5), E2 generates Acetyl-CoA:

Acetyl-dihydrolipoamide + CoA → Acetyl-CoA + dihydrolipoamide
*Acetyl-CoA = entry point of the CAC

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7
Q

What are the mechanistic advantages of multi-enzyme complexes?

A
  1. Minimized distances for substrates in between active sites → increases reaction rate without having to maintain large pool of intermediates
  2. Metabolic intermediates are channeled between successive enzyme sites → minimization of side reaction + protection for chemically labile intermediate
  3. Coordinated controls of reactions → shutting off one enzyme effectively shuts the system down
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8
Q

How is the Pyruvate Dehydrogenase reaction regulated?

A

*ALL E1
By allosteric product inhibition
Accumulation of NADH and Acetyl-CoA shut down E1 through product inhibition → prevents useless consumption of pyruvate

By phsphorylation of E1 in the PDC

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9
Q

How does PDC regulation by phosphorylation occur?

A

Inhibition:
E1-CH2OH + ATP → (PDH kinase) → E1-PO4(2-) + ADP
- Activated by ATP, NADH, Acetyl-CoA (activation of the inhibition)
- Inhibited by pyruvate, ADP

Activation:
E1-PO4(2-) + H2O → (PDH phosphatase) → E1-OH + Pi
- Activated by presence of Mg2+, Ca2+, insulin

*By phosphorylation of 3 specific serine residues

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10
Q

Does the CAC consume O2?

A

No, it is part of the aerobic metabolism, but does not direclty consume O2

The CAC can run without O2, but the cell would run out of NAD and FAD+

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11
Q

Is the CAC present in prokaryotes and eukaryotes?

A

No, only in aerobic metabolism of eukaryotes

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12
Q

When and by who was discovered the CAC?

A

1937

  1. Albert Szent-Györgyi → respiration
  2. Hans Krebs → CAC
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13
Q

Summarize the CAC.

A

The CAC is a series of 8 enzymatic reactions that combine acetyl CoA (2 C) with oxaloacetate (4 C) to generate CO2, NADH, FADH2 and regenerates the starting product oxaloacetate.

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14
Q

What does it mean for the CAC to be amphibolic?

A

It is a site of anabolism and catabolism

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15
Q

What part of the CAC corresponds to anabolism and catabolism?

A

Anabolism → CAC intermediates are the starting point of anabolic pathways (ex: gluconeogenesis, fatty acid synthesis, amino acid synthesis)

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

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16
Q

What is a Cataplerotic reaction vs an anaplerotic reaction?

A

Cataplerotic (cata = emtpying) → depletes the CAC intermediates → decreases cycle chain

Anaplerotic (ana = filing up) → replenish the depleted CAC intemediates

*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

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17
Q

What are the main function of the CAC?

A
  1. Producing reducing equivalents
  2. Produce intermediates for biosynthesis
  3. Produce ATP
18
Q

Does the CAC harvest energy?

A

Yes, through electric gradients

19
Q

What is the overall reaction of the CAC?

A

3 NAD+ + FAD + GDP + Pi + acetyl-CoA → 3 NADH + FADH2 + GTP + CoA + 2CO2

20
Q

What are the first and last reactions of the CAC?

A

1st: Oxaloacetate + Acetyl-CoA + H2O → (citrate synthase) → Citrate (+ CoASH)

Last (8th): Make oxaloacetate again to be able to restart the cycle
L-Malate + NAD+ → (malate dehydrogenase) → oxaloacetate + NADH + H+
*Take 2 H+ off

21
Q

What CAC intermediate does the CO2 come from?

A

Step 4:
a-ketoglutarate + CoA-SH + NAD+ → (a-ketoglutarate dehydrogenase)→ Succinyl-CoA + CO2 + NADH + H+

H from CoA-SH is replaced by a-ketoglutarate which loses its COO- (becomes the CO2)
*Very similar to PDC

In step 5, CoASH is remade and energy of breaking the Succinyl-CoA thioester bond allows to make GTP from GDP + Pi
Succinyl-CoA + GDP → (succinyl-CoA synthetase) → Succinate + CoASH + GTP

22
Q

What is the net energy production / cycle of the CAC?

A

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

23
Q

How is the whole CAC regulated?

A

Regulated by the main ∆G negative steps → steps 1, 3 and 4 (non-reversible steps)
*Activators = metabolites upstream to be used by the CAC
*Inhibitors = produced by the CAC, accumulating when too much CAC

Step 1:
- Inhibited by NADH, Succinyl-CoA

Step 3:
- Activated by ADP, Ca2+
- Inhibited by NADH, ATP

Step 4:
- Activated by Ca2+
- Inhibited by Succinyl-CoA, NADH

24
Q

What factors activate/inhibit the transformation of Pyruvate to Acetyl-CoA (PDC)?

A

Activators:
- Mg2+, Ca2+
- Insulin
- ADP
- Pyruvate

Inhibitors:
- Acetyl-CoA
- NADH
- ATP

25
Q

What is the CAC flux responsive to?

A
  1. Energy state of the cell through allosteric activation (ex: Isocitrate dehydrogenase by ADP)
  2. Redox state of the cell through the mitochondrial NADH/NAD ratio
  3. Availability of energy rich compounds (acetyl-CoA, succinyl-CoA) that inhibits CAC Enzymes (CS and alpha-KGDH)
26
Q

What are the values for the total Aerobic metabolism balance sheet ?

A
  1. Glycolysis (cytosol):
    2 ATP → 2 ATP
    2 NADH (2.5 ATP/NADH) → 5 ATP
    Total: 7 ATP/glucose
  2. Pyruvate dehydrogenase (mitochondria):
    1 NADH (2.5 ATP/NADH) → 2.5 ATP
    Total: 2.5 ATP/pyruvate → 5 ATP/glucose
  3. CAC (mitochondria):
    3 NADH (2.5 ATP/NADH) → 7.5 ATP
    1 FADH2 (1.5 ATP/FADH2) → 1.5 ATP
    1 GTP (1 ATP/GTP) → 1 ATP
    Total: 10 ATP/acetyl-CoA → 20 ATP/glucose

For every glucose → 32 ATP

27
Q

In the micochondria, between the intermembrane space and the matrix, which one is more positively charged/contains the protons?

A

Protons are pumped from the matrix → the intermembrane space (positively charged)

NADH+ H+ are in the matrix, ADP/ATP are in the matrix, O2/H2O are in the matrix

28
Q

What are the roles of the different mitochondrial complexes of the inner mitochondrial membrane?

A

Complexes I → Oxidizes NADH + H+ to NAD and takes the electrons to pump 4H+
Complex III → pumps 4H+
Complex IV → pumps 2H+

*Conformational changes in the complexes as electrons pass through them allow them to pump H+ from matrix to the intermembrane space

NOT complex II → tranfers electrons from FADH2 to CoQ which brings them to complex III
Complex II is also a succinate dehydrogenase (SDH) which catalyses conversion of succinate to fumarate, while doing FAD → FADH2

29
Q

What is the role of Coenzyme Q in the electron transport chain?

A

It acts as an electron carrier by taking electrons from Complex I and II → complex III

30
Q

What is the role of Cyt c in the electron transport chain?

A

Shuttles electrons between complex III and IV of the inner mitochondrial membrane

31
Q

How many ATP molecules are made for every NADH and every FADH2 in the electron transport chain?

A

1 NADH → 2.5 ATP (enters the chain at complex I so pumps 10H+)
1 FADH2 → 1.5 ATP (enters the chain at complex II so pumps 6 H+ instead of 10H+)

*1 NADH + H+ or 1 FADH2 = 2 electrons/2H+ = 1 H2O = 1/2 O2

32
Q

What is the voltage gradient across the mitochondrial inner membrane?

A

150-200mV ~ 30 million Volts/meter

33
Q

How much energy is released from the reaction of the electron transport chain making H2O out of NADH?
What is the complete reaction?

A

NADH + H(+) + 1/2 O2 → NAD(+) + H2O
∆G = -220 kJ/mol

*Gives a lot of energy to make ATP ~ 32kJ/mol

34
Q

Do electron flow from high to low or from low to high reduction potential?

A

They flow from LOW → HIGH reduction potential (towards more and more negative ∆G)

NADH > 0 reduction potential as it will give electrons
O2 < 0 reduction potential as it will accept electrons

Reduction potential = how much a molecule wants to accepts or donate electrons

35
Q

Who is responsible for the chemi-osmotic theory?

A

Peter Mitchell

36
Q

What are the evidence that the chemical-osmotic coupling theory is right? (and not the high intermediate theory for the electron transport chain) (6)

A
  1. The respiratory chain can function in absence of phosphate
  2. The # moles of ATP generated through NADH oxidation was nto an integer (for high intermediate, it would have to be)
  3. An intact inner mitochondrial membrane (IMM) is required for OXPHOS
  4. Key electron transport proteins are all localized in the IMM
  5. Uncouplers (2,4-Dinitrophenol) DNP inhibit ATP synthesis → they allow protons to cross the membrane freely
  6. Generating an artificial proton gradient permits ATP synthesis without electron transport (its not the proteins, its really the gradient)
37
Q

In mammals, how many C subunits form the F0 complex?

A

8 C subunit on the c-ring of F0→ for every 360˚ turn → 8 protons enter (1/c subunit of the c-ring)

38
Q

How many ATP molecules are synthesized for every 360˚ turn of the F0 complex?

A

3 ATP molecules produced by F1 for every turn F0:
8 H+ → 3 ATP molecules

39
Q

How many protons are pumped for every single ATP molecules synthesized?

A

For 1 ATP:
- 8 H+/3ATP → 2.7H+/1ATP
- 1H+ for Pi symport to the matrix

Totals of 3.7H+/ATP molecules

40
Q

What is the proton to oxygen ratio for NADH? And the resulting ATP production for NADH.

A

NADH: (10H+ for every 2 electrons)/(3.7 H+/ATP molecule) ~ 2.5 ATP/NADH

  • 10 H+due to the H+ pumped across complex I (4H+), complex III (4H+), complex IV (2H+)
  • 3.7 H+/ATP molecule → 8H+ from ATP synthase ~ 3 ATP
  • For every produced in the matrix, 1 Pi is pumped with an H+ → 3 additional H+
    11 H+ for 3 ATP produced → 11/3 → 3.7 H+/ATP produced

Total → 10 H+/NADH ÷ 3.7 H+/ATP ~ 2.5 ATP/ NADH

41
Q

What is the proton to oxygen ratio for FADH2? And the resulting ATP production for FADH2.

A

NADH: (6H+ for every 2 electrons)/(3.7 H+/ATP molecule) ~ 2.5 ATP/NADH

  • 6 H+due to the H+ pumped across complex III (4H+), complex IV (2H+)
  • 3.7 H+/ATP molecule → 8H+ from ATP synthase ~ 3 ATP
  • For every produced in the matrix, 1 Pi is pumped with an H+ → 3 additional H+
    11 H+ for 3 ATP produced → 11/3 → 3.7 H+/ATP produced

Total → 6 H+/NADH ÷ 3.7 H+/ATP ~ 1.5 ATP/ NADH

42
Q

Why can we count the NADH produced in the cytosol from glycolysis in the total ATP count?

A

Because the protons are transported to the electron transport chain through shuttling proteins that will reduce NAD similarly as the CAC to form further NADH in the mitochondria → ETC.

BIOC 212 (15 decks)

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