TCA cycle

Question 1

Define the TCA cycle and ATP production

Answer 1

Pyruvate produced by oxidation of glucose is further oxidized via the citric acid cycle to produce ATP

Question 2

What does the complete oxidation of pyruvate to CO2 and H2O require?

Answer 2

Oxygen

Question 3

Aerobic catabolism is also called?

Answer 3

Cellular respiration

Question 4

Where does Cellular respiration occur?

Answer 4

In most Eukaryotic cells and Bacteria

Question 5

What are the 3 major phases of cellular respiration?

Answer 5

1) Production of Acetyl-CoA
2) Oxidation of Acetyl-CoA to form CO2 and reduced coenzymes
3) Oxidation of reduced coenzymes via the electron transport chain

Question 6

What are the first 2 major steps for the catalytic mechanism for pyruvate dehydrogenase?

Answer 6

1) Pyruvate is decarboxylated: Formation of hydroxyethyl-thiamine pyrophosphate
2) Formation of acetyl-lipoic acid by oxidation of lipoic acid disulphide: TPP is regenerated by this reaction

Question 7

What are the 3rd and 4th major steps for the catalytic mechanism for pyruvate dehydrogenase?

Answer 7

3) Acetyl group transfer from acetyl-lipoic acid to form acetyl-CoA
4) Re-oxidation of reduced dihydrolipoamide formed in reaction 3: Enzyme is prepared for another round of oxidative decarboxylation

Question 8

TCA cycle is amphibolic, define Amphibolic

Answer 8

Serves as energy source and supplies intermediates to the biosynthetic pathways

Question 9

Define the TCA cycle being under stringent control

Answer 9

The entry of acetyl-CoA and metabolically irreversible steps are control points

Question 10

How is pyruvate dehydrogenase regulated?

Answer 10

Variety of different means:

Allosteric activation by pyruvate, NAD+ and CoA-SH

Allosteric inhibition by ATP, NADH and acetyl-CoA
Allosteric inhibition is enhanced by presence of long chain fatty acids

Question 11

What is the role of Pyruvate dehydrogenase kinase

Answer 11

Phosphorylation of E1 (inactivates).

Allosterically activated by ATP

Question 12

What is the role of Pyruvate dehydrogenase phosphatase?

Answer 12

Dephosphorylation of E1 (activates)

When kinase is allosterically inhibited, phosphatase re-activates pyruvate dehydrogenase

Question 13

TCA cycle, step 1: What happens to Oxaloacetate?

Answer 13

Citroyl-CoA is a transient intermediate formed on the active site

Large negative free energy change of thioester hydrolysis favours the formation of citrate

An essential feature of reaction, since mitochondrial [oxaloacetate], is low

CoA-Sh liberated is recycled for further rounds of pyruvate decarboxylation of Beta-oxidation

Question 14

What enzyme catalyses the formation of citrate?

Answer 14

Citrate synthesis

Question 15

TCA cycle, step 2: What happens to Citrate?

Answer 15

Isomerisation of citrate by Aconitase

Tertiary alcohol of citrate is a poor candiate for further oxidation

Results in C-C bond cleavage

Isomerisation to secondary alcohol resolves this problem

Aconitase promotes reverisble addition of H2O to cis-aconitate

Isomerisation of citrate to isocitrate is endergonic:
Less than 10% isocitrate at equilibrium under standard conditions
Formation of isocitrate under cellular favoured by isocitrate consumption in step 3 of TCA cycle

Question 16

TCA cycle, step 3: What happens to Isocitrate?

Answer 16

Oxidative decarboxylation: Sufficiently exergonic to isomerisation in favour of isocitrate formation

2 step reaction:

1) Oxidation of C2 alcohol of isocitrate (formation of oxalosuccinate)
2) beta-decarboxylation. Beta-keto acids are unstable and readily decarboxylate

First link between TCA cycle, electron transport chain and biosynthetic pathways:

Alpha-ketoglutarate is an important substrate for aminotransferase reactions

Question 17

What catalyses the formation of Cis-aconitate and Isocritate?

Answer 17

Aconitase

Question 18

What catalyses the formation of Alpha-ketoglutarate?

Answer 18

Isocitrate dehydrogenase

Question 19

TCA cycle, step 4: What happens to Alpha-ketoglutarate?

Answer 19

Forms Succinyl-CoA

Alpha-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2) and dihydrolipoyl dehydrogenase (e3) form a tri-functional enzyme

E3 is conserved between the 2 complexes

Free energy of decarboxylation and oxidation of Alpha-ketoglutarate is conserved in high energy thioester linkage

Second link between TCA cycle, electron transport and biosynthesis

Succinyl CoA and NADH are important sources of metabolic energy for cellular processes

Key regulatory step in the TCA cycle

Question 20

What catalyses the formation of Succinyl-CoA?

Answer 20

Alpha-ketoglutarate dehydrogenase

Question 21

TCA cycle, step 5: What happens to Succinyl-CoA?

Answer 21

Forms Succinate

Energy of phosphorylation provided by substrate rather than proton gradient/electron transport chain

Free energy of hydrolysis of succinyl-CoA and ATP (or GTP) are similar

ATP is formed directly in plants and bacteria: In animals, GTP is converted to ATP by nucleotide diphosphate kinase

Question 22

What catalyses the formation of Succinate?

Answer 22

Succinyl-CoA synthetase

Question 23

TCA cycle, step 6: What happens to Succinate?

Answer 23

C=C bond formation to make Fumarate

Oxidation of C-C to C=C not sufficiently exergonic to reduce NAD+
Can only reduce FAD

Complex quaternary structure
Forms part of the succinate-coenzyme Q reductase of the electron transport chain

Required for both catalysis and electron transport

Associated with the inner mitochondrial membrane

Question 24

What catalyses the formation of Fumarate?

Answer 24

Succinate dehydrogenase

Question 25

TCA cycle, step 7: What happens to L-Malate?

Answer 25

Trans-hydration of fumarate to form Malate

Fumarase is an example of prochiral enzyme

Non-chiral, but enzyme only produces L-isomer of Malate

Trans-addition of H2O across the double bond

Question 26

What catalyses the formation of L-Malate?

Answer 26

Fumarase

Question 27

TCA cycle, step 8: What happens to Malate?

Answer 27

Completion of the cycle: Dehydrogenation of Malate

Keq of reaction favours of Malate over oxaloacetate

Reaction is driven towards oxaloacetate formation by maintaining the intra-mitochondrial [oxaloacetate] very low.

Continuous utilisation of oxaloacetate by citrate synthase helps to achieve this

Loss of Oxaloacetate to other pathways can rapidly shut down TCA cycle unless levels are replenished