Pyruvate Dehydrogenase

Question 1

What does PDH stand for?

(In the context of enzymes)

Answer 1

Pyruvate dehydrogenase

Question 2

What role does the PDH complex serve?

(In an overall sense)

Answer 2

It act as a link between glycolysis and the TCA cycle by converting pyruvate into acetyl-CoA so that it may pass into the mitochondrial matrix and feed into the cycle.

Question 3

Identify:

The specific overall reaction carried out by PDH.

Answer 3

Oxidative decarboxylation of pyruvate to form acetyl-CoA.

This generates an NADH as well. (2 NADH overall, as left with 2 pyruvate at the end of glycolysis).

Question 4

List:

The THREE enzymes that make up the pyruvate dehydrogenase (PDH) complex.

Answer 4

• E1: Pyruvate dehydrogenase.
• E2: dihydrolipoyl transacetylase.
• E3: dihydrolipoyl dehydrogenase.

Question 5

Which binding domains (BD) comprise the pivot point for the lipoyl domain (LpD) ‘swinging arms’?

Answer 5

E1BD & E3BD

Question 6

How many reactions and coenzymes contribute to the oxidative decarboxylation of pyruvate by the PDH complex?

Answer 6

Five reactions and coenzymes.

Note: CoA-SH and NAD⁺ act as substrates too.

Question 7

Describe:

The ΔG°’ of the PDH catalysed reaction, and what this indicates.

Answer 7

It is large and negative, indicating that it is spontaneous and irresversible.

Specifically, it is -33.4 kJ mol^-1.

Question 8

List:

The FIVE coenzymes of the PDH complex.

Answer 8

• TPP (thiamine pyrophosphate; from vitamin B₁)
• Lipoic acid
• CoA (coenzyme A; from vitamin B₅)
• FAD (flavin adenine dinucleotide; from vitamin B₂)
• NAD⁺ (nicotinamide adenine dinucleotide; from vitamin B₃)

Question 9

What is the role of the LpD (lipoyl domain) ‘swinging arms’ in the PDH complex?

Answer 9

To deliver electrons and intermediates from one active site to another.

Question 10

What part of the PDH complex is the coenzyme TPP bound to, and by what kind of interaction?

Answer 10

Non-covalently bound to E1.

Question 11

What part of the PDH complex is the coenzyme lipoic acid bound to, and by what kind of interaction?

Answer 11

Covalently bound to a Lys on E2.

This forms a lipoamide.

Question 12

What part of the PDH complex is the coenzyme CoA bound to, and by what kind of interaction?

Answer 12

Non-covalently bound to E2.

It is a substrate of E2 and forms part of the product (acetyl-CoA) of the PDH complex.

Question 13

What part of the PDH complex is the coenzyme FAD bound to, and by what kind of interaction?

Answer 13

Covalently bound to E3.

Question 14

What part of the PDH complex is the coenzyme **NAD^{+** bound to, and by what kind of interaction?}

Answer 14

Non-covalently bound to E3.

It is a substrate of E3, and is converted to NADH.

Question 15

Why can thiamine deficiency lead to severe neurological symptoms?

Answer 15

The brain exclusively utilises aerobic respiration to generate energy.

Thiamine is not stored nor synthesised well in most vertebrates, and so must be obtained via diet.

Question 16

What disease is caused by chronic thiamine deficiency?

Answer 16

Beri Beri disease

Main symptoms include neurological dysfunction, paralysis, limb atrophy, and cardiac failure.

Question 17

What happens when lipoic acid binds with Lys on E2 and E3BP?

Answer 17

It forms a long lipoamide arm that can swing between the active sites of E1, E2, and E3.

Question 18

What is unique or important about the disulfide bond of the lipoamide arm?

Answer 18

It may be reduced to a dithiol group, which can then act as an electron carrier or acyl carrier.

It undergoes redox reactions for these roles.

Question 19

How does arsenic alloesterically inhibit the PDH complex?

Answer 19

It readily and irreversibly binds to the dithiol group of the lipoamide arms.

Question 20

What is important about the thiol group of CoA?

(Thiol = a carbon-bonded sulfhydryl ; C-SH)

Answer 20

It can covalently bind with an acyl group to form a thioester bond.

This has a high hydrolysis potential (i.e. large negative ΔG), and essentially ‘activates’ the molecule with the acyl group.

An example is acetyl-CoA.

Question 21

What acts as the electron acceptor in the E3 reaction of the PDH complex?

Answer 21

NAD⁺

Question 22

Define:

Prosthetic group

(in the context of enzymes)

Answer 22

A non-amino acid component that covalently binds to enzymes and assists in their function.

For example, FAD covalently binds to E3 of the PDH complex.

Question 23

Identify:

The enzyme(s), substrate, end product, and any other important byproducts/reactants of the PDH complex reaction I.

Answer 23

• E1: Pyruvate dehydrogenase
• Decarboxylation of pyruvate to a 2C acetyl group.
• Binding of this acetyl group to TPP to form a reactive hydroxyethyl-TPP.

Note: TPP first binds to E1 before reacting with the pyruvate.

CO₂ is the byproduct of this step.

Question 24

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the PDH complex reaction II.

Answer 24

• E1: pyruvate dehydrogenase to E2: dihydrolipoyl transacetylase
• A redox reaction whereby the hydroxyethyl-TPP of E1 is oxidised as the hydroxethyl is transferred to the lipoamide disulphide arm of E2 (reduction) to form a thioester intermediate (acetyl-dihydrolipoamide).

Question 25

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the PDH complex reaction III.

Answer 25

• E2: dihydrolipoyl transacetylase
• Acetyl group transferred to CoA by E2.
• Simultaneously, the lipoamide is reduced (dihydrolipoamide).

Question 26

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the PDH complex reaction IV.

Answer 26

• E2: dihydrolipoyl transacetylase to E3: dihydrolipoyl dehydrogenase
• Redox reaction where the dihydrolipoamide arm is oxidised by reducing the FAD prosthetic group of E3.

Note: in this step, the hydrides attached to FAD are then transferred to the disulfide of the E3 dihydrolipoyl (thus reducing it). This is the disulfide interchange.

Question 27

Identify:

The enzyme, substrate, end product, and any other important byproducts/reactants of the PDH complex reaction V.

Answer 27

• E3: dihydrolipoyl dehydrogenase
• NAD⁺ is reduced by FADH₂ after it has reoxidised the disulfide of E3.

In this FAD is an important intermediate.

Question 28

What is the disulfide interchange during step IV of the PDH reaction complex?

Answer 28

When the FADH₂ passes its two hydrides to the disulfide of the E3 dihydrolipoyl temporarily.

Question 29

What are the starting substrates and end products of the PDH complex reaction?

Also known as the transition or prepatory stage for the TCA cycle.

Answer 29

• SS: 2 pyruvate + 2 CoA-SH + 2 NAD⁺
• EP: 2 acetyl-CoA + 2 NADH

Note: TPP, FAD, and lipoate are also vital for the process.

Question 30

List:

FOUR key advantages of multi-complex enzymes.

(Like the PDH complex)

Answer 30

• Higher reaction rate.
• Reduction of diffusion dependency.
• Reduced side reactions.
• Multiple regulation sites + coordinated regulation.