citric acid cycle (Krebs cycle)

what is the citric acid cycle?

series of vital biochemical reactions in cellular respiration that cells use to release stored energy

“A cyclic pathway for the oxidation of acetyl residues to carbon dioxide, in which formation of citrate is the first step”

what is the purpose of the citric acid cycle?

convert stored energy into chemical energy that can be used

where does the citric acid cycle occur and what conditions does it require?

• takes place in mitochondrial matrix

• requires oxygen

what are the three main categories of macromolecules in human diets?

• proteins (amino acids)

• carbohydrates (sugars)

• fats (fatty acids)

what does the breakdown of macromolecules produce?

• Energy

• Reducing power (reduced coenzymes)

• Useful carbon (can be used to make other compounds)

general outline of the citric acid cycle

• oxidises acetyl CoA (derived from proteins, carbs and fats) into carbon dioxide and water

• energy is released as ATP, NADH and FADH₂

how are carbohydrates/sugars broken down to acetyl CoA?

through glycolysis

• sugars → pyruvate → acetyl CoA

how are fatty acids broken down to acetyl CoA?

through fatty acid oxidation

how are amino acids broken down to acetyl CoA?

amino acid catabolism

what compounds are released during the citric acid cycle?

• reduced coenzymes

• carbon dioxide

• GTP

• reduced coenzyme Q (interaction with ETC)

what is the link reaction?

links cystolic glycolysis (anaerobic) to the citric acid cycle (mitochondrial metabolism (aerobic))

occurs in the mitochondrial matrix

describe the link reaction process

pyruvate (from cytosol) enters the mitochondrial matrix

pyruvate is decarboxylated and oxidised by pyruvate dehydrogenase (enzyme) to form acetyl CoA

what type of reaction is the link reaction?

oxidative decarboxylation

link reaction diagram

why is the link reaction important?

• important for regulation

• once pyruvate is converted to acetyl CoA it cannot be converted back - IRREVERSIBLE

• pyruvate can be used to make glucose but acetyl CoA cannot - important to maintain pyruvate during fasting/starvation

what is the rate of the link reaction in fed and fasting states?

• fed state: link reaction occurs at high rates

• fasting/starvation: link reaction does not occur (pyruvate dehydrogenase enzyme is inhibited)

step 1 of citric acid cycle?

• oxaloacetate (4C) + acetyl CoA (2C) → citrate (6C)

• thioester bond in acetyl CoA is hydrolysed - exothermic so provides enough energy to form C-C bond with oxaloacetate

• reaction is very exothermic so enzyme is regulated

• ΔG = -32kJ/mol

what enzyme is used to hydrolyse thioester bond/synthesise citrate?

citrate synthase

step 2

• citrate (6C) → isocitrate (6C)

• citrate is rearranged into an isomer of itself

• standard ΔG° = +13.3kJ/mol

• actual ΔG in cell = 0

how is reaction catalysed if there is a +ΔG°?

• the reaction before and after this (step 1/3) are very exothermic

• enzyme is receiving lots of substrate from the step 1 reaction and products are being quickly removed by step 3 reaction

what enzyme is used in step 2?

aconitase (type of isomerase)

step 3

• isocitrate (6C) → alpha-ketoglutarate (5C)

• NADH (NAD → NADH) and CO₂ also produced

• oxidative decarboxylation

• ΔG° = -20.9kJ/mol (very exothermic)

• favourable process as taking a reduced product that would rather be oxidised

what enzyme is used in step 3?

isocitrate dehydrogenase (remove H, oxidise substrate)

enzyme is regulated

step 4

• alpha-ketoglutarate → succinyl CoA

• NADH and CO₂ also produced

• oxidative decarboxylation

• ΔG° = -33.5kJ/mol (very exothermic)

what enzyme is used in step 4?

alpha-ketoglutarate dehydrogenase

regulated enzyme

step 5

• succinyl CoA → succinate

• GTP or ATP is also produced (GDP/ADP → GTP/ATP)

• thioester bond in succinyl CoA is hydrolysed which provides energy for substrate level phosphorylation

• ΔG° = -2.9kJ/mol (similar energy is provided by hydrolysis as is used for phosphorylation)

• reversible reaction

what enzyme is used in step 5?

succinyl CoA synthetase

• two versions of the enzyme: version 1 ADP → ATP, version 2 GDP → GTP

• version used depends on what the cell needs

step 6

• succinate → fumarate

• (coenzyme Q → coenzyme QH₂ also happens)

• oxidation reaction, double bond formation

• ΔG° = 0

what enzyme is used?

succinate dehydrogenase

• only enzyme in the cycle bound to the IMM

• enzyme is part of the electron transport chain, same as complex II

• electrons from succinate are transferred directly to ETC

step 7

• fumarate → L-malate

• hydration reaction, addition of H₂O

• highly stereospecific reaction, OH group on malate on left hand side

• ΔG° = -3.8

what enzyme is used in step 7?

fumarase (type of lysase enzyme)

step 8

• L-malate → oxaloacetate

• (NAD → NADH)

• ΔG° = +29.7

• oxidation reaction

• oxaloacetate is regenerated and the cycle repeats

what enzyme is used in step 8?

malate DH

how is reaction catalysed with positive ΔG°?

reaction is pulled forwards by the exothermic reaction that follows (STEP 1)

ETC, succinate dehydrogenase and reduction potential

• Reduction potential of succinate DH/complex II is very similar to reduction potential of coenzyme Q

• When succinate transfers electrons to coenzyme Q, NO PROTONS are pumped because succinate does not have negative enough reduction potential

• only complex III and IV pump protons

• substrates reduced by flavoproteins have lower P:O ratio becaus less ATP is made per oxygen reduced

• only 6H⁺ pumped when succinate DH is used to reduce oxygen

what is the overall yield of the oxidative catabolism of 1 mole of acetyl CoA

• 3 NADH

• 2 CO₂

• 1 FADH₂

• 1 GTP/ATP

• 1 CoA

why is the citric acid cycle an aerobic pathway?

ETC is used which indirectly uses oxygen

needed to turn NADH back into NAD and coenzyme QH₂ into coenzyme Q

what cells does the citric acid cycle occur in?

all cells

how does the rate of the citric acid cycle change in different metabolic states?

the cycle is NOT AFFECTED by metabolic state

metabolic state does affect where acetyl CoA is obtained

where is acetyl CoA obtained from in the fed state?

glycolysis increases so acetyl CoA comes from sugars

where is acetyl CoA obtained from in the fasting state?

acetyl CoA comes from fatty acids because glycolysis is inhibited

where is acetyl CoA obtained from in the exercise state?

acetyl CoA supply is increased from fatty acids and glucose

how is citric acid cycle activity increased?

• Increase activity of enzymes - (citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase - regulated enzymes)

• Increase supply of acetyl CoA

• Increase concentration of intermediates - e.g. oxaloacetate