Pyruvate Dehydrogenase complex Flashcards
What are the enzymes that make up the pyruvate dehydrogenase complex?
E1, E2 and E3.
What are the cofactors used by the pyruvate dehydrogenase complex?
TPP, lipoamide, CoA, FAD and NAD.
What reaction does the pyruvate dehydrogenase complex catalyse?
The decarboxylation of pyruvate to form acetyl-CoA (the link reaction).
Why can a β keto acid be decarboxylated without a cofactor?
The carbanion intermediate can be stabilised by resonance structures as the keto group is on the adjacent carbon- keto/enol tautomerisation.
Why can an α-keto acid not be decarboxylated without a cofactor?
It cannot form resonance structures in order to stabilise the carbanion intermediation, therefore requiring a cofactor to act as an electron sink, e.g. TPP is used by pyruvate dehydrogenase.
What are the 3 main stages of the conversion of pyruvate to Acetyl-CoA by pyruvate dehydrogenase?
- Decarboxylation
- Oxidation
- Transfer of intermediate to CoA
How does the pyruvate dehydrogenase complex allow the turnover of many pyruvate reactions at once?
The complex has multiple copies of each enzyme, with E1 on the outside, and E2/E3 on the inside.
How does TPP stabilise the carbanion intermediate?
By covalent catalysis, nucleophilic attack of the α-keto group by TPP.
What is the overall mechanism of the pyruvate dehydrogenase complex?
Pyruvate forms carbanion, and is covalently attached to TPP. Carbanion is transferred to fully oxidised state of lipoamide. Nucleophilic attack of the keto group by CoA. E2 oxidised to reform lipoamide. Disulfide bridge in E2 is reduced. FAD is reduced to regenerate disulfide bridge. FADH is then used to reduce NAD.
What is the E1 mechanism of pyruvate dehydrogenase?
Base in the active site removes a proton to form the carbanion. Nucleophilic attack of the α-keto by TPP. Hydroxylethyl-TPP intermediate is stabilised by resonance structures.
How can a disulfide bridge be broken?
By the addition of 2 electrons or by the addition of a carbanion.
How does the lipoamide swinging arm assist catalysis?
The lipoamide is flexible to pick up acetyl from any E1 subunits. Allows electron transfer between lipoamides on different E2 subunits until the electrons reach an acetyl group to perform the transferase reaction with.
What is the E2 mechanism?
Nucleophilic attack of the lipoamide arm by the carbanion, forming a sulfide. Base in the active site removes a proton from the intermediate, reforming C=O. Nucleophilic attack by CoASH forms acetyl-CoA.
What reaction does E2 catalyse?
CoA + Acetyllipoamide => Acetyl-CoA + Dihydrolipoamide
- this is reversible
- the transfer step in the overall pyruvate to acetyl-CoA conversion
What is the E3 mechanism?
Hydride transfer reactions to regenerate lipoamide and FAD.
- from lipoamide to FAD
- from FADH to NAD (via movement of electrons in the ring structure of FADH)
