sakai-PDH and TCA cycle

Question 1

How is pyruvate transported from the cytosol into mitochondria? Where in the cell are the PDH complex and the TCA cycle performed?

Answer 1

Pyruvate in the cytosol is transported by a pyruvate carrier across the inner mitochondrial membrane. In this process pyruvate enters together with H+ in exchange for OH- .

The PDH complex and the TCA cycle are both performed inside of mitochondria.

Question 2

The pyruvate dehydrogenase complex (PDH) in mitochondria allows the connection of aerobic glycolysis with the TCA cycle. What is the overall reaction?

Answer 2

Pyruvate and free CoA (HSCoA) and NAD+ are used to form acetyl CoA and NADH and carbon dioxide.

Question 3

Describe the actions of enzyme 1, 2 and 3 of the PDH complex.

Answer 3

Enzyme 1 decarboxylates pyruvate to a molecule that is used as substrate for enzyme 2.
Enzyme 2 uses HSCoA and forms acetyl CoA and carbon dioxide.
Enzyme 3 uses NAD+ and forms NADH.

Question 4

What are the coenzymes (cofactors) of the individual enzymes of the PDH complex? Are they also needed in other pathways? Summarize!

Answer 4

E1 needs thiamine-PP (from vitamin B1) .
E2 needs lipoic acid. It uses HSCoA and forms acetyl CoA.
E3 needs FAD and uses NAD+ and forms NADH.

Question 5

What coenzyme is needed for TCA (alpha keto glutarate DH), PPP(transketolase), and PDH(enzyme 1)?

Answer 5

Thiamine-PP

Question 6

when Thiamine-PP is absent what difiecency occurs?

Answer 6

Deficiency in Wernicke-Korsakoff and in Beri-Beri

Thiamine-PP is formed from vitamin B1.

Question 7

what coenzyme is needed for PDH: enzyme 2,

TCA: -ketoglutarate DH?

Answer 7

Lipoic acid

Question 8

what is special about lipolic acid?

Answer 8

Inhibition by trivalent arsenic (arsenite)

Question 9

what coenzyme is needed for PDH: enzyme 2

TCA: -ketoglutarate DH,-oxidation of fatty acids?

Answer 9

HSCoA

Question 10

what is special about HSCoA?

Answer 10

Acetyl CoA and succinyl CoA contain energy rich bonds

Question 11

what coenzyme is needed for PDH: enzyme 3

TCA: -ketoglutarate DH, and succinate DH?

Answer 11

FAD

Question 12

what is sepcial about FAD?

Answer 12

FAD is a prosthetic group and is tightly bound to the enzyme.

FADH2 leads in the ETC and oxidative phosphorylation to 2 (1.5) ATP

Question 13

what coenzyme is needed for Glycolysis: glyceraldehyde 3-P DH
PDH: enzyme 3
TCA:  isocitrate DH
          -ketoglutarate DH
          malate DH?

Answer 13

NAD+ is a free coenzyme found in cytosol and mitochondria

NADH leads in the ETC and oxidative phosphorylation to 3
(2.5) ATP

Question 14

PDH is an important link between aerobic glycolysis and the TCA. How is it regulated in general terms?

Answer 14

PDH is product inhibited by acetyl CoA and NADH.
Calcium ions stimulate PDH. They activate PDH phosphatases.

NADH is formed in E3, and acetyl CoA is formed in E2.
[Acetyl CoA and NADH formed by -oxidation and the NADH generated in the TCA cycle also lead to inhibition of PDH.]

Question 15

PDH is also inhibited by phosphorylation by a specific PDH kinase. How is this PDH kinase allosterically regulated?

Answer 15

PDH kinase phosphorylates and inhibits PDH at enzyme 1 (pyruvate decarboxylase).
PDH kinase is allosterically activated by acetyl CoA and NADH and ATP.
PDH kinase is allosterically inhibited by pyruvate.

That means that when ATP is high, and acetyl CoA and NADH are high, the PDH should not be used to form acetyl CoA and pyruvate shall be saved.

Question 16

When do we want to activate PDH? Describe the action of calcium in regulation of pathways! Discuss the TCA cycle and glycogen degradation!

Answer 16

The PDH shall be active during muscle contraction when glycogen degradation and aerobic glycolysis lead to pyruvate that can be used as acetyl CoA in the TCA cycle.

In general, calcium ions activate
glycogen phosphorylase kinase using the calmodulin subunits,
the PDH complex (indirect via activation of PDH phosphatase)
and the TCA at the level of isocitrate DH and -ketoglutarate DH.

Question 17

Are PDH kinase and PDH phosphatase mainly regulated by hormones or are they mainly allosterically regulated?

Answer 17

They are mainly allosterically regulated by the energetic demands inside of the cell.

Calcium ions activate PDH phosphatases and leads to an active PDH complex.
(Insulin may act via enzyme induction of PDH phosphatases)

Question 18

Describe PDH regulation in the liver!

Answer 18

In the liver, PDH has to be tightly regulated, as after pyruvate is used to form acetyl CoA, the carbons of the acetyl-group cannot be used for gluconeogenesis.

In the liver, pyruvate is saved for gluconeogenesis when needed.

The PDH complex is inhibited by acetyl CoA and NADH generated by -oxidation during fasting.

[Pyruvate carboxylase is used for gluconeogenesis and forms oxaloacetate. It needs the absolute activation by acetyl CoA which is formed during -oxidation of fatty acids]

Question 19

Which enzyme of the TCA cycle needs the same coenzymes as the PDH complex? Name other similarities!

Answer 19

The TCA cycle enzyme -ketoglutarate DH needs the same coenzymes as the PDH complex.
Both are inhibited by NADH and both are activated by calcium ions, but in a different way.

Both catalyze a decarboxylation, use HSCoA, and form NADH.

PDH forms acetyl CoA, TCA cycle forms succinyl CoA.

Special for PDH and not found for ketoglutarate DH is that PDH is also regulated by PDH kinase and PDH phosphatase.

PDH kinase inhibits PDH and PDH kinase is activated by acetyl CoA and NADH.
PDH phosphatase activates PDH and is itself activated by calcium ions

Question 20

Describe congenital lactic acidosis. Which enzyme of the PDH complex is deficient?

Answer 20

Enzyme 1 is deficient and the pyuvate is used to form lactate.
Symptoms include neurodegeneration, muscle spasticity and can lead to early death.

Question 21

In case of poisoning with trivalent arsenic (arsenite), which coenzyme is bound by arsenite and inhibited? Which enzyme of the PDH complex is inhibited? Which enzyme of the TCA cycle is inhibited?

Answer 21

Arsenite forms a stable complex with lipoic acid.

Lipoic acid it used in E2 of the PDH complex, and also by -ketoglutarate DH

Question 22

Compare poisoning with trivalent arsenic to the poisoning with pentavalent arsenic (arsenate). Which enzyme of glycolysis uses arsenate instead of inorganic phosphate?

Answer 22

Trivalent arsenic (arsenite) binds to lipoic acid. 
Pentavalent arsenic (arsenate) competes with inorganic phosphate. 

Arsenate is used instead of inorganic phosphate by glyceroaldehyde 3-phosphate dehydrogenase.
Instead of the formation of 1,3-bisphosphoglycerate, a now instable molecule is formed which immediately leads to 3-phosphoglycerate instead of 1,3-bisphosphoglycerate.
With that, one of the two steps of substrate level phosphorylation in glycolysis cannot be performed normally

Question 23

Discuss the consequences of thiamine deficiency (Wernicke-Korsakoff and Beri-Beri) related to PDH, TCA cycle and PPP.

Answer 23

Brain is dependent on energy generation by aerobic glycolysis, PDH and TCA cycle.

Thiamine-PP is needed for the PDH and -ketoglutarate DH. This means that deficiency of thiamine-PP leads to less generation of ATP.

In cells with high levels of NADPH, the oxidative part of the PPP cannot be used to form ribose-5-P. Transketolase fulfills the important function to generate ribose-5-P (together with transaldolase in the reversible part of the PPP) .

Transketolase is two times involved and needs thiamine-PP as coenzyme. The deficiency of thiamine-PP leads to less formation of ribose 5-P in cells that cannot use the oxidative part of the PPP.

Question 24

Explain the central role of the TCA cycle related to catabolism and anabolism.

Answer 24

The TCA cycle has catabolic functions including two steps of decarboxylations.
It is also able to generate 3 NADH and 1 FADH2 and 1 GTP.

On the other hand, the TCA cycle has also anabolic function, for example related to synthesis of amino acids or of heme (succinyl CoA) . It also provides metabolites from glucogenic amino acids which can be used for gluconeogensis in the liver.

Question 25

What is the purpose of the TCA cycle related to its catabolic function? Tell the story!

Answer 25

In the TCA cycle we want to decarboxylate so that the carbon atoms can be exhaled as carbon dioxide and we want to generate reductive equivalents for ATP generation.

[note: decarboxylation takes place in the PDH and PPP, but not in glycolysis.]

Citrate synthase uses oxaloacetate and acetyl CoA in order to form citrate.
[The TCA cycle is reduced in its activity, when oxaloacetate is used for gluconeogenesis in the liver.]

Citrate cannot be decarboxylated and isocitrate is formed by aconitase.
[Aconitase is inhibited by fluoroacetate/fluorocitrate]

Isocitrate dehydrogenase decarboxylates isocitrate to -ketoglutarate and forms NADH.

-ketoglutarate dehydrogenase(complex) decarboxylates and forms succinyl CoA and NADH.

[Note that both allosterically regulated enzymes of the TCA cycle are the steps of decarboxylation.]

Now the purpose of decarboxylations is fulfilled, and in order to finish the cycle, an effective way to form again oxaloacetate is performed which generates GTP, FADH and one more NADH.

The enzyme succinate thiokinase uses succinyl CoA and GDP and forms succinate and GTP. The enzyme succinate dehydrogenase forms fumarate and FADH2 [ succinate dehydrogenase is competitively inhibited by malonate]. The enzyme fumarase forms malate and finally malate dehydrogenase forms oxaloacetate and NADH.

Question 26

Why is the TCA cycle inhibited by lack of oxygen? What are the effects of fluorocitrate and malonate?

Answer 26

The TCA cycle needs NAD+ and FAD. Lack of oxygen inhibits the ETC and NADH and FADH accumulate. And with that, NAD and FAD are not available for the TCA cycle.

Fluorocitrate and malonate inhibit the TCA cycle and can lead to cell death.

Fluorocitrate is formed from the poison fluoroacetate and inhibits aconitase. [Fluoroacetate is found in plants and also in pesticides, especially “1080”]

Malonate is a structural analog of succinate and competitively inhibits succinate dehydrogenase. This inhibition was used to investigate the catalysis of succinate dehydrogenase.

Question 27

Which enzymes catalyze irreversible steps of the TCA and how are these enzymes regulated?

Answer 27

Citrate synthase catalyzes an irreversible step and starts the TCA cycle. This enzyme in humans is mainly regulated by substrate availability (oxaloacetate and acetyl CoA) which activates it and on the other hand citrate synthase is product inhibited by citrate.

The main allosteric regulated enzymes of the TCA cycle in humans are isocitrate DH and -ketoglutarate DH. These enzymes catalyze irreversible reactions, both decarboxylate and generate NADH and carbon dioxide.

Isocitrate DH is inhibited by NADH and ATP,
-ketoglutarate DH is inhibited by NADH and succinyl CoA.

Both enzymes are directly activated by calcium ions.

Question 28

Why is aerobic metabolism of glucose using the TCA cycle much more efficient than anaerobic glycolysis? Describe the concept.

Answer 28

The following shows the concept and it is calculated that NADH leads to 3 ATP and that FADH2 leads to 2 ATP. (if 2.5 and 1.5 are used, the total numbers change a lot)

Glycolysis from glucose leads to two pyruvates and generates 2 ATP and 2 NADH.
The NADH when used in the malate-aspartate shuttle leads to 6 ATP.
So far, 8 ATP are generated from aerobic glycolysis.

The two pyruvates are used in the PDH complex which generates 2 NADH and 2 acetyl-CoAs. The 2 NADH generate 6 ATP. So far, we generated totally 14 ATP.

The 2 acetyl CoAs are used in the TCA cycle and lead to 6 NADH, 2 FADH2 and 2 GTP. This adds up to 22 ATP and 2 GTP. Totally we have now 36 ATP and 2 GTP.
Sometimes GTP is seen as ATP equivalent and this leads to 38 ATP in total.

It is clear that the combination of aerobic glycolysis with PDH and the TCA cycle leads to efficient ATP generation by substrate level phosphorylation and to even more ATP formation by oxidative phosphorylation.

Question 29

⦁ In the TCA, succinyl CoA is cleaved to succinate. How can the cleavage of this high energetic bond be used?

Answer 29

Succinyl CoA is an energy-rich molecule and is cleaved to succinate in the TCA.
The energy generated is used to link GDP and Pi and form GTP.

This is the step of substrate level phosphorylation of the TCA cycle.