Tricarboxylic Acid (TCA) Cycle

Tricarboxylic acid (TCA) Cycle / Citrate Cycle

“hub” of cellular metabolism; captures energy using redox reactions

Functions of TCA cycle

Primary function: To oxidize acetyl-CoA

1. Generates the bulk of NADH and FADH2, which are used to produce ATP by oxidative phosphorylation

2. Links the oxidation of metabolic fuels (carbohydrates, fatty acids, proteins) to ATP through shared intermediates

3. It provides metabolites for other biosynthetic pathways (fatty acids, amino acids, hemes)

Metabolic pathways compartmentalization

Glycolysis takes place in the cytoplasm

Citric acid cycle takes place in the mitochondrial matrix

Oxidative phosphorylation takes place along inner mitochondrial membrane

pyruvate dehydrogenase

Pyruvate + CoA + NAD = Acetyl-CoA + CO2 + NADH + H+

ΔG° = -33.4 kJ/mol

Contains 3 distinct catalytic enzymes (E1-E3)

Uses 5 different cofactors or co-enzymes

Essentially irreversible (commits pyruvate to aerobic respiration)!

pyruvate dehydrogenase complex

pyruvate dehydrogenase (E1) - decarboxylation w/ TPP

dihydrolipoyl transacetylase (E2) - transfer to CoA

dihydrolipoyl dehydrogenase (E3) - oxidation of lipoamide

Why use a complex?

Catalytic sites are close to one another, which allows channeling of substrates

Avoids side reactions with intermediates

Facilitates coordinated regulation of activity of different subunits

Pyruvate dehydrogenase steps

1. Decarboxylation - attaches to TPP cofactor, lose CO2!

2. Activation of Ac - thioester formed; hydroxyethyl group is oxidized/ reacts with disulfide of lipoamide attached to Lys of E2, which acts as the oxidant and is itself reduced

3. Transthioesterification - Acetyl-CoA production; occurs in active site of E2 with CoA; dihydrolipoamide is now fully reduced

4. Oxidation of dihydrolipoamide - transfers 2e-, 2 H+ to E3 disulfide, which then gets re-oxidized and reduces FAD to FADH2

5. Oxidation of FADH2 to FAD by NAD+ to produce NADH + H+ - regenerates FAD

Net Reaction of one turn of TCA cycle

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O → CoA + 2 CO2 + 3 NADH + 3 H+ + FADH2 + GTP

TCA cycle electron transfer

Each “turn” of citrate cycle produces 4 pairs of e- (8 e-)

History of TCA cycle

Krebs described the cycle in 1937.

Krebs and Henseleit also discovered urea cycle (urea synthesized by amino acids and ammonia)

Lipmann discovered role of acetyl-CoA in metabolism (early 1940s)

Krebs and Lipmann share 1953 Nobel Prize in Physiology.

TCA Cycle steps

Aerobic pathway

Continuous (cyclic)

Eight reactions

Naming the cycle

TCA cycle products and carbon backbones

Lipoamide

extracts the acetyl group from TPP

Lipoamide transfer mechanism

Lipoamide regeneration

regenerated by oxidation with FAD, which transfers e- to NAD+

dihydrolipoyl transacetylase “ball and chain” mechanism

arsenite

dihydrolipoyl transacetylase inhibitor; covalent binding to reduced lipoamide

Coenzyme A (CoA)

common “acyl carrier compound”; half deprotonated at physiological pH

Citrate synthase

OAA + aectyl-CoA + H2O → citrate + CoA

Why is citrate synthase reaction so favorable? (ΔG°’ = -31.4 kJ/mol)

CoA release - breaking thioester bond

aconitase

Fe-S cluster facilitates removal of OH group from citrate

fluoroacetyl-CoA

can be turned into flurocitrate → inhibits aconiase activity and blocks citrate export

isocitrate dehydrogenase

first oxidative decarboxylation of the cycle (and first production of NADH)

oxidation helps stabilise decraboxylation

alpha-ketoglutarate dehydrogenase complex

Second oxidative decarboxylation of the cycle, which also produces NADH

similar to pyruvate dehydrogenase

succinyl-CoA synthetase

Goes through high energy intermediates (succinyl-phosphate and phospho-His)

succinate dehydrogenase

This is complex II in the ETC (direct link!)

Produces FADH2 (used directly in ETC)

fumarase

hydration rxn

malate dehydrogenase

Redox reaction

Produces final NADH!

decarboxylation reactions

lead from 6-carbons to 4-carbon molecules

biochemical standard free energy changes for TCA reactions

Why is this high ΔG°′ tolerated?

citrate synthase very favourable + low concentrations of OAA

TCA cycle regulation points

pyruvate dehydrogenase

pyruvate carboxylase

citrate synthase

isocitrate dehydrogenase

alpha-ketoglutarate dehydrogenase

TCA cycle regulation

Product inhibition by acetyl-CoA, NADH, ATP, other carbon products

excepy pyruvate carboxylase, which is allosterically upregulated by acetyl-CoA

ADP regulation

can activate enzymes by relieving allosteric inhibition by ATP

Ca2+ regulation

signals need for ATP during muscle contraction

Regulation of pyruvate dehydrogenase (PDH) by PTM

regulated by phosphorylation; relates to cellular energy charge

E1 is phosphorylated by KDH kinase/ dephosphorylated by the phosphatase

Kinase is activated by NADH, acetyl-CoA, ATP to block PDH function; NAD+/CoA/ADP inhibit kinase