Energy generation in mitochondria L3

Question 1

What are the 3 enzymes that take part in the formation of acetylCoA from pyruvate?

Answer 1

1. Pyruvate Dehydrogenase (with Thiamine Pyrophosphate as a substrate)
2. Dihydrolipoyl transacetylase (w/ lipoic acid covalently bound)
3. Dihydrolipoyl DH (w/ FAD covalently bound)

NOTE: And FADH2→FAD makes NADH from NAD+

Question 2

What inhibits and activates pyruvate DH?

Answer 2

Products: AcetylCoA and NADH inhibit, as well as GTP;

Activated by AMP

Question 3

How is pyruvate DH modified for inhibition/activation?

Answer 3

Phosphorylation of a specific serine on pyrDH leads to INACTIVATION. This is done by Mg²⁺ kinase, which is activated by ATP, NADH, Acetyl CoA, and inhibited by pyruvate (ie pyr allows PDH to stay active);

A phosphatase will dephosphorylate PDH and to activate it and it is activated by Ca²⁺ ions

Question 4

How is α-Ketoglutarate DH a similar complex to the PDH complex?

Answer 4

The first 2 enzymes are similar for αKG DH (ie similar to PDH and dihydrolipoyl transacetylase),

and the 3rd enzyme is the same (dihydrolipoyl DH)

Question 5

How does gluconeogenesis affect malate transport?

Answer 5

It causes malte to leave the mitocondrial matrix via the malate transporter enzyme b/c malate is a major substrate for glc synthesis (malate→OAA→Glc).

IN FACT, ALL C₄ acids are reduced in mitochondria during gluconeogenesis b/c formation of pyr from glc is also reduced under these conditions.

(so TCA cycle slows down)

Question 6

By what is pyruvate carboxylase activated by and why?

Answer 6

Pyruvate Carbyoxylase uses biotin and CO₂ to make oxaloacetate (OAA) from pyruvate. This is activated by Acetyl CoA b/c in an abundance of AcetylCoA, OAA is made so TCA cycle can begin.

Question 7

What is the overal rxn equation for the TCA cycle?

Answer 7

AcCoA+ 3NAD+ FAD+ GDP + P_i → 2CO₂+ 3NADH+ FADH₂+ GTP+ CoA

Question 8

Describe the rxns and enzymes of the entire TCA cycle.

Where does the TCA cycle take place?

Answer 8

Takes place in the matrix:

1. AcCoA+OAA→Citrate+CoA [citrate synthase]
2. Citrate→isocitrate [aconitase]
3. Isocitrate+NAD⁺→αKG+NADH+CO₂ [Isocitrate DH]
4. αKG+NAD⁺+CoA→SuccinylCoA+NADH+CO₂ [αKG DH]
5. SuccinylCoA+GDP+P_i→Succinate+GTP+CoA [succinate thiokinase]
6. Succinate+FAD→fumarate+FADH₂ [Succinate DH]
7. Fumarate+H₂O→Malate [Fumarase]
8. Malate+NAD⁺→OAA+NADH [Malate DH]

Question 9

How are citrate synthase and isocitrate DH regulated?

Answer 9

Citrate Synthase is inhibited by ATP;

Isocitrate DH is inhibited by ATP, and activated by ADP

Question 10

What is the overall yield of ATP from a single turn of the TCA cycle?

Answer 10

3NADH, 1FADH₂, 1GTP= 12ATP

Question 11

What is the only enzyme of the TCA cycle not located in the mitochondrial matrix?

Answer 11

Succinate DH, which is located on the IMM facing the matrix