Citric acid cycle

Question 1

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

Answer 1

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

Question 2

How does pyruvate enter the mitochondrion?

Answer 2

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

Question 3

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

Answer 3

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

Question 4

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

Answer 4

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)

Question 5

How does step 5 of the pyruvate dehydrogenase reaction occur?

Answer 5

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

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

Question 6

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

Answer 6

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

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

Question 7

What are the mechanistic advantages of multi-enzyme complexes?

Answer 7

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

Question 8

How is the Pyruvate Dehydrogenase reaction regulated?

Answer 8

*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

Question 9

How does PDC regulation by phosphorylation occur?

Answer 9

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

Question 10

Does the CAC consume O2?

Answer 10

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+

Question 11

Is the CAC present in prokaryotes and eukaryotes?

Answer 11

No, only in aerobic metabolism of eukaryotes

Question 12

When and by who was discovered the CAC?

Answer 12

1937

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

Question 13

Summarize the CAC.

Answer 13

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.

Question 14

What does it mean for the CAC to be amphibolic?

Answer 14

It is a site of anabolism and catabolism

Question 15

What part of the CAC corresponds to anabolism and catabolism?

Answer 15

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

Question 16

What is a Cataplerotic reaction vs an anaplerotic reaction?

Answer 16

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

Question 17

What are the main function of the CAC?

Answer 17

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

Question 18

Does the CAC harvest energy?

Answer 18

Yes, through electric gradients

Question 19

What is the overall reaction of the CAC?

Answer 19

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

Question 20

What are the first and last reactions of the CAC?

Answer 20

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

Question 21

What CAC intermediate does the CO2 come from?

Answer 21

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

Question 22

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

Answer 22

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

Question 23

How is the whole CAC regulated?

Answer 23

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

Question 24

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

Answer 24

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

Inhibitors:
- Acetyl-CoA
- NADH
- ATP

Question 25

What is the CAC flux responsive to?

Answer 25

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)

Question 26

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

Answer 26

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

Question 27

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

Answer 27

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

Question 28

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

Answer 28

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

Question 29

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

Answer 29

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

Question 30

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

Answer 30

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

Question 31

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

Answer 31

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

Question 32

What is the voltage gradient across the mitochondrial inner membrane?

Answer 32

150-200mV ~ 30 million Volts/meter

Question 33

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

Answer 33

NADH + H(+) + 1/2 O2 → NAD(+) + H2O ∆G = -220 kJ/mol *Gives a lot of energy to make ATP ~ 32kJ/mol

Question 34

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

Answer 34

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

Question 35

Who is responsible for the chemi-osmotic theory?

Answer 35

Peter Mitchell

Question 36

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

Answer 36

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)

Question 37

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

Answer 37

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

Question 38

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

Answer 38

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

Question 39

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

Answer 39

For 1 ATP: - 8 H+/3ATP → 2.7H+/1ATP - 1H+ for Pi symport to the matrix Totals of 3.7H+/ATP molecules

Question 40

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

Answer 40

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

Question 41

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

Answer 41

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

Question 42

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

Answer 42

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.