Bio98 Lecture 14 TCA Cycle

Know the different reactions and enzymes in the TCA cycle (substrates mnemonic)

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Pyruvate, Citrate, Isocitrate, α-Ketoglutarate, Succinyl-CoA, Succinate, Fumarate, Malate, Oxaloacetate

TCA Rxn 1: Acetyl-CoA Condensation w/ OAA

IRREVERISIBLE RXN: Aldol condensation (H2O release).

• Oxaloacetate/OAA (4C) + acetyl-CoA (2C) → regenerates free CoA and Citrate (6C)

• Enzyme: carried out by citrate synthase + release of H2O

• Acetyl-CoA forms the enolate

TCA Rxn 2: Isomerization of Citrate

REVERSIBLE RXN:

COMBINED DEHYDRATION-HDYRATION RXN: isomerizes tertiary alcohol citrate to secondary alcohol isocitrate

-Citrate (prochiral) turns into isocitrate (stereospecific rxn produces only one isomer) through aconitase (enzyme); basically just relocates the OH in diff positions

Fluoroacetate (1080) poison

Fluorocitrate is a suicide substrate for aconitase ; Australian “poison peas”

TCA Rxn 3: Oxidation of isocitrate to α-KG

IRREVERSIBLE RXN:

Summary: the secondary alcohol in isocitrate (6C) is oxidized (dehydrogenated) to give the intermediate oxalosucciante, which decarboxylates to give α-KG or α-ketoglutarate (5C)

-carried out by ISOCITRATE DEHYDROGENASE (IDH)

-uses NAD+ to release 1st NADH/H+ & 1st CO2 released

• removal of a CO2 (decarboxylation rxn)

-regulated by substrate availability and product inhibition

TCA Rxn 4: α-KG oxidative decarb. to succinyl-CoA

IRREVERSIBLE RXN:

Summary: α-ketoglutarate undergoes oxidative decarboxylation, a concerted rxn in which the carboxyl group closest to the carbonyl is lost as CO2 and succinyl-CoA is formed.

-1) α-ketoglutarate (5C) undergoes oxidative decarboxylation bc of α-ketoglutarate dehydrogenase ( KGDH enzyme) → NAD+ is required = release of 2nd NADH/H+

-2) Succinyl-CoA (4C) and 2nd CO2 made

Nomenclature: α-KG is oxidized, NAD+ is reduced. When something is oxidized, something else is reduced

TCA Rxn 5: Succinyl-CoA to succinate

REVERSIBLE RXN:

-oxidation & substrate level phosphorylation rxn; rxn that directly generates ATP or GTP.

-Thioester provides energy for phosphorylation.

• since GDP/ADP are getting phosphorylated (ie the substrate) rather than protein, that’s termed as substrate level phosphorylation.

-1) Succinyl-CoA (4C) undergoes rxn bc of succiynl-CoA synthetase (enzyme); GDP or ADP + Pi + H2O make GTP or ATP

-2) Succinate(4C) + HS-CoA are also made

TCA Rxn 6: Oxidation of succinate to fumarate

REVERSIBLE RXN: (THE ARROW SHOULD BE REVERSIBLE)

Summary: in this oxidation, e-s from succinate are transferred to FAD to form the trans dioic acid fumarate

-1) Succinate (4C) undergoes rxn bc of succinate dehydrogenase; FAD accepts electrons, gets reduced, oxidizes C-C bonds, and makes FADH2

-2) Fumarate (4C) gets made

-All the other TCA enzymes are soluble in matrix but his onte is found in the inner mitochondrial membrane (close to ETC)

TCA Rxn 7: Hydration of fumarate to malate

REVERSIBLE RXN:

Summary: the alkene moiety of fumarate is hdyrated to form the secondary alcohol in malate

• Fumarate (4C) undergoes hydration (H2O) + fumarase (enzyme) = Malate (4C)

Fumarase Deficiency

-Autosomal recessive, leads to physical and neurological abnormalities 1:400 million chance in “the wild”

-Super rare, ~100 cases total worldwide, almost entirely within the FLDS community Genetic disorders impacting central metabolism (TCA) are extremely rare because of its importance to life

TCA Rxn 8: Oxidation of Malate to Oxaloacetate

REVERSIBLE RXN:

Summary: the hydroxyl group of malate is oxidized to the carbonyl of oxaloacetate. The e-s are harvested to NAD+ in the process.

-1) Malate (4C) undergoes rxn bc of malate dehydrogenase & NAD+ → release of 3rd NADH/H+

-2) Makes low oxaloacetate (4C)

Which are the irreversible steps? Do they have neg. or positive ΔG?

Rxn 1 (citrate synthase), 3 (IDH), 4 (KGDH); have largest negative overall ΔG

TCA Recap and Final Numbers

• Overall, we have produced:

• 3 NADH

• 1 FADH2

• 1 ATP (or GTP)

• 2 CO2

• Per molecule of glucose, TCA makes:

• 6 NADH

• 2 FADH2

• 2 ATP (or GTP)

Glycolyiss + PDH + TCA total

TOTAL: 4xATP + 10xNADH + 2xFADH2 per glucose up to this point

-Glycolysis- Net 2xATP + 2Xs Pyruvate + 2xNADH per glucose (uses 2 ATP but generates 4)

-PDH- 1xNADH per pyruvate (2x per glucose)

-TCA- 1xATP + 3xNADH + 1xFADH2 per acetyl-CoA (2x per glucose)

Anaplerotic rxn or “anaplerosis”

Molecules that are synthesized out of the TCA cycle can often be converted back into TCA intermediates

• In pancreatic cancer cells, glutamine provides nearly all the carbon that goes through the TCA cycle