Tca

Front 1

List the reactions and products of the many "fates" of pyruvate in metabolism.

What cellular conditions determine how pyruvate will partition into these reactions?

Back 1

1. Pyruvate Decarboxylase: CO2 + Acetaldehyde-->Alcohol DH + NADH + H+: EtOH + NAD+

Lactate DH + NADH-->Lactate + NAD+

Pyruvate DH Complex: AcetylCoA+ CO2-->further oxidation

Pyruvate Decarb & Latctate DH during anaerobic conditions (muscle).
AcetylCoA during aerobic conditions.

Front 2

Why are anaerobic pathways important for glycolysis?

Why doesn't this happen under aerobic conditions?

Back 2

NADH converted back into NAD by anaerobic pathways (using up NAD in glycolysis and then glycolysis produces NADH). If glycolysis stops, then you get no energy.

Anaerobic pathways allow recylcing of NADH to NAD+ so glycolysis can occur.

In TCA you go from NAD-->NADH but oxidative phospho takes the NADH and converts it back to NAD. So any NADH produced under aerobic conditions goes to mito for oxidative phospho.

Front 3

What reaction does pyruvate DH catalyze? What kind of reaction is it and what side product is released?

Back 3

Oxidizes pyruvate and attaches to CoA to form Acetyl-CoA, releases CO2.

Front 4

Which carbon is lost from Acetyl CoA? How many rounds of TCA are required to remove all carbons contributed by AcetylCoA?

Back 4

Check Slide 1 of TCA. 2 cycles are needed.

Front 5

What is the general reaction catalyzed by DH enzymes?

Back 5

Oxidation

Front 6

Why do radioactive carbons from Fatty Acids end up in glucose is you can't use fat to make glucose?

Back 6

Fats broken down into Acetyl CoA, after one cycle of TCA, one carbon from acetylCoA kept, so you have a carbon from the fat in oxaloacetate, which is a precursor to gluconeogenesis.

Front 7

What three steps (reactive) are used to get from pyruvate to acetylCoA?

What enzyme catalyzes these steps and what are its 'subareas' (and their functions)?

Back 7

Decarb, Oxidation, Transfer acetyl to CoA

PDH: E1 does decarb and oxidizine, E2 does transfer to CoA, E3 gives electrons to FAD

Front 8

What is the importance of E3 in PDH?

Back 8

Since electrons are going into the enzyme, the enzyme must regenerate itself by getting rid of these electrons. E3 does this by giving up these electrons to FAD.

Front 9

What are the prosthetic groups of each subunit of PDH?

Back 9

E1: TPP
E2: Lipoamide
E3: FAD

Front 10

Thoroughly describe the mechanism of PDH.

Back 10

Pyruvate enters PDH and is bound to TPP in E1, release of CO2.

Lipoamide 'arm' extends from E2 into E1 who oxidizes TPP and brings acetyl group into E2.

CoA enters the enzyme and the acetyl group is transferred to CoA.

The reduced lipoic acid group then enters E3 where it is oxidized by FAD to return to its original state.

NAD+ enters to replace NADH and the enzyme is ready for another round of dehydration.

Front 11

What is the main benefit of having a big multi-enzyme complex like PDH?

Back 11

Since all the active sites are near one another, hydrolysis by water of intermediates is prevented (by water). It's efficient.

Front 12

What is the function of citrate synthase? What byproducts are released?

Back 12

Combines acetylCoA with oxaloacetate to form Citryl CoA and then Citrate!

CitrylCoA --> Citrate is a hydrolysis reaction (releases CoA)

Front 13

Describe the mechanism of citrate synthase.

Back 13

Oxaloacetate interacts with a his, while an Asp acts as a base and removes H from Acetyl-CoA, causing a second His to act as an acid and donate an H to Acetyl-CoA, this forms the high energy ENOL intermediate.

His then acts as base to remove H from Enol causing Acetyl-CoA to accak oxaloacetate and remove H from Histidine. This is the citryl-CoA complex.

Hydrolysis of the thioester of the CoA drives the reaction forward and pushes the equil to citrate formation (otherwise would get equilibrium and go back to substrate form)

Front 14

How is a-Ketoglutarate DH similar to pyruvate DH? Why does this make sense?

Back 14

Homologous E1, E2, identical E3.

E1 recognizes reactant and then it's connected to lipoamid arm in E2. Because E3 catalyzes the same reaction (oxidation of lipoic acid to give FADH2), it can be exactly the same.

E1 and E2 are catalyzing similar reactions.

Front 15

Why doesn't succinyl CoA Synthetase require a 'great' nucleophile to drive its reaction forward? Describe its mechanism.
What reactions are being coupled?

Back 15

Succinyl CoA has high energy thioester. So phosphate is used a nucleophile to attack thioester and give CoA.
Still have a high energy intermediate P-Succinyl-CoA, so histidine will attack phosphate (phosphorylated his) and succinate will be released.

GDP then enters and his phosphorylates the GDP to form GTP.

Coupling the hydrolysis of a thioester to form a phosphoanhydride bond.

Front 16

What other TCA enzymes share the same mechanism of action as PDH?

Back 16

Fumarase, Malate DH, Succinate DH, a-Keto DH

Front 17

Why is PDH so highly regulated?

How is it regulated and what inhibits/activates it?

Back 17

Pyruvate can undergo many reactions, can be used to make aa's, gluconeogenesis, form acetylCoA, fatty acid synthesis, etc. It's a very important branchpoint.
High concentrations of reaction products of the complex inhibit the enzyme: acetylCoa inhibits E2, NADH inhibits E3.

Phosphorylation of E1 by a kinase turns of the complex and its deactivation is reversed by the action of a specific phosphatase. The site of phosphorylation is in E2 (emphasizing importance of this core).

High energy charge and abundance of biosynthetic precursors (lots of NADH and AcetylCoA and ATP) will activate Kinase activity and inhibit PDH.
Pyruvate, ADP, NAD+ inhibit the kinase. Ca++ increases activity of the phosphatase.

Front 18

What are the three control points of TCA? Why are they control points? What are the inhibitors/activators of each step (activators when relevant)?

Back 18

Pyruvate-->AcetylCoA, Isocitrate to a-keto, a-keto to Succinyl CoA.
These are branchpoints. It's where cells are making a decision: spend a-ketoglutarate to make protein or food (ATP)?

Pyr-->Acetyl: -: ATP, acetylCoA, NADH

Iso-->a-keto: -: ATP, NADH, +: ADP

a-keto-->succinyl: -: ATP, succinyl CoA, NADH