PDC

Question 1

Where does the PDC reaction occur?
How does pyruvate get into the mitochondrion?

Answer 1

In the mitochondrion
Pyruvate is already transported from the cytoplasm to the mitochondrion through the pyruvate translocase (H+ symport)

*Also ADP/ATP antiport

Question 2

What general reaction is catalyzed by the Pyruvate dehydrogenase?

Answer 2

Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH

Question 3

What is the structure/components of the PDC?

Answer 3

Size = 9.5 MegaDalton in eukaryotes
(compared to pyruvate which is 88 Daltons)

E1 → 30 heterotetramers/complex
E2 → 60 core monomers/complex
E3 → 12 homodimers/complex
E3-binding protein → 12
Pyruvate dehydrogenase kinase → 1-3
Pyruvate dehydrogenase phosphatase → 1-3

Question 4

What are the 5 different coenzymes required to function with the PDC?

Answer 4

1) TPP → bound to E1: decarboxylate pyruvate → hydroxyethyl-TPP + CO2
2) Lipoic acid → covalently linked to Lys on E2: accepts hydrozyethyl from TPP
3) CoA → Substrate for E2: accepts acetyl group from lipoamide
4) FAD → bound to E3: Reduced by lipoamide
5) NAD+ → substrate for E3: Reduced by FADH2

Question 5

How many reactions occur in the PDC?
How many Enzymes?

Answer 5

5 reactions, 3 enzymes

Question 6

What are the mechanistic advantages of a Multienzyme Complexe such as PDC?

Answer 6

1. Minimized distances for substrates in between active sites → increases reaction rate without having to maintain large pools of intermediates
2. Metabolic intermediates are channeled between successive enzye sites → Side reactions are minimized + protection for chemically labile intermediates
3. Coordinated control for reactions/regulation → shutting of 1 enzyme effectively shuts the system down

*Overall more efficient

Question 7

What is the role of E1 in the PDC?

Answer 7

E1 = Pyruvate Dehydrogenase → Dearboxylation reaction
Pyruvate + TPP-E1 → CO2 + Hydroxyethyl-TPP-E1
*TPP is always bound to E1
- Nucleophilic attack by Thiamine Pyrophosphatase → decarboxylation → C1 of pyruvate released as CO2
- C2 and C3 attached to TPP

Question 8

What makes the whole PDC irreversible?

Answer 8

Step 1 because CO2 diffuses out of the mitochondria as a gas (can’t use it as a substrate) → Step 1 is irreversible (all other steps are reversible)

Question 9

What occurs in step 2 of the PDC reaction?

Answer 9

TPP (bound to E1) is regenerated as the hydroxyethyl group is passed onto Lipoamide (bound to E2) → Acetyl-dihydrolipoamide

→ The hydroxyethyl group is oxidized to acetic acid. The 2 electrons removed in oxidation reduce the S-S of the lipoyl group on E2 → SH
→ Makes a thioesther high energy bond

*Reversible

Question 10

What are the 2 products of E2 in step 2 and 3?

Answer 10

Step 2 regenrates TPP for E1
Step 3 generates acetyl-CoA

Question 11

What happens in step 3 of the PDC?

Answer 11

Trans-esterification to CoA:
Acetyl-dihydrolipoamide + CoA → Acetyl-CoA + reduced form of Lipoamide (dihydrolipoyl)

Lipoamide is E2’s “swinging arm” to perform substrate channeling between E1 and E3

Question 12

What is the function of CoA?

Answer 12

Functions as a carrier of acetyl and other acyl groups

Question 13

What are the characteristics of Acetyl-CoA?

Answer 13

• High energy thioester bond between acetyl group and CoA
• High energy compound → hydrolysis = -31.5kJ/mol
• Has a high acyl group transfer potential and can donate the acetyl group to several acceptors
• Entry point of CAC

Acetyl group → O=C(~S)-CH3

Question 14

What is the role of E3 in the PDC? (Explain steps 4 and 5)

Answer 14

*Step 4 and 5 are reversible
Goal: Reset E2 and E3 to their active state

Step 4: Oxidation-reduction reactions → generate oxidized E2-lipoamide (reform disulfide bond on lipoamide, by reducing the on on FAD

Step 5: enzyme-bound FADH2 reoxidized to FAD by NAD+ → NADH → Ox. Phos. (re-activate E3 that was deactivated in step 4)

Question 15

Why is regulation of the Pyruvate Dehydrogenase Complex essential?

Answer 15

No other pathway in mammals for synthesis of acetyl-CoA from pyruvate

Animals can’t synthesize glucose from acetyl-CoA so conversion of pyruvate → acetyl-CoA commits glucose carbons to oxidation in CAC or fatty acid synthesis

Question 16

What are the 2 levels of control of the the PDC?

Answer 16

1. Product inhibition by acetyl-CoA and NADH
2. Covalent modification (phosphorylation of E1 pyruvate dehydrogenase)

Question 17

How does product inhibition work at a molecular level?

Answer 17

CoA comes into E2 active site and is converted to Acetyl-CoA which fits in the active site as well and then goes away
Since acetyl-CoA (product) also fits in the active site and the enzyme if reversible, if it accumulates, Acetyl-CoA will go to the active site as a substrate and the reverse reaction will occur

Step 1 is the same, but since CO2 diffuses out of the mitochondria, it will not go into the active site for the reverse

Question 18

What happens in the PDC when [acetyl-CoA] and [NADH] are high?

Answer 18

*ALLOSTERIC REGULATION
1. Reactions 3 and 5 run backwards
2. E2 is in the acetylated form and does not proceed forward
3. E3 is reduced (SH) when high NADH and cannot regenrate S-S in E2

→ Accumulation of acetyl-CoA and NADH shuts down E1 through product inhibition (TPP not regenerated by E2)
→ Prevents useless consumption of pyrvate

Question 19

How is PDC COVALENTLY regulated? (simple)

Answer 19

PDK phosphorylates E1 to inactivate it → shuts down entire PDC
PDP dephosphorylates E1 to activate it

Question 20

What allosteric regulators modulate the activity of PDK and PDP?

Answer 20

Acetyl-CoA and NADH → allosteric activators of PDK (inhibitor of PDC)
pyruvate, ADP, Ca2+ (high Mg2+), K+ → allosteric inhibitors of PDK

Mg2+, Ca2+ → allosteric activator of PDP (activate PDC)
*Ca2+ released when muscle contraction activate PDC → make more ATP (logical)

Question 21

Which 2 enzyme activities regulate the utilization of pyruvate in the cell?

Answer 21

PDC (→ Acetyl-CoA) and LDH (→ lactate)
*In cancer cells, PDC activity is suppressed by phosphrylation through PDK → pyruvate is converted to lactate (Warburg effect)

Question 22

What happens to the PDC when you stop running?

Answer 22

1. No need to produce ATP → Acetyl-CoA and NADH accumulate
2. Causes E2 and E3 to turn OFF by product inhibition
3. E1 is OFF through phosphorylation

Question 23

What happens to the PDC when you start running?

Answer 23

1. Need to produce ATP
2. Activated glycolysis produce pyruvate → blocks PDK and activates E1 (allosterically)
3. Ca2+ increases for muscle contraction → activates PDP → E1 turned ON

*All reactions in multienzymes complex are tightly regulated