BCH 333 Chapter 17: Citric Acid Cycle

Citric Acid Cycle

• complete oxidation of carbon to CO2

• Harvests electrons

• 2 CO2, 8 electrons, 1 GTP/ATP

• PE from electrons cannot be used directly —> later used to generate a proton gradient which can be used to do work

Pyruvate oxidation

• key irreversible step (can’t go back to glucose/gluconeogenesis) (in animals—plants and some bacteria can convert acetyl CoA to glucose via the glyoxylate cycle)

• Pyruvate —> acetyl CoA

• Occurs in the lumen/mitochondrial matrix (pyruvate is imported via a pyruvate transporter in the IMM)

• Acetyl CoA can also be used for fatty acid synthesis (mt and cytosol) or can be generated by fatty acid degradation in the mitochondrial matrix

• Regulated via allosteric interactions and phosphorylation

• Catalyzed by the PDC

Pyruvate dehydrogenase complex (PDC) regulation

• downregualted by high energy charge (NADH, acetyl CoA, ATP)

• Deactivated by phosphorylation

• Reactivated by phosphatase

• Upregulated by low energy charge (pyruvate, ADP)

Pyruvate dehydrogenase complex (PDC)

• three enzymes that catalyze multiple steps in the reaction pathway within the same complex

• Pretty large (larger than ribosomes)

• Catalytic cofactors: TPP, lipoid acid, FAD

• Stoichiometric cofactors (function as substrates): CoA and NAD+

• 3 steps—decarboxylation, oxidation, and transfer to CoA

• Minimizes side reactions, maximizes rate, and allows for the coordinated catalysis of reactions

• E1 adds 2C to TPP —> E2 takes 2C from TPP, adds to CoA —> E3 re-oxidizes E2 lipoamide, reducing FAD/NAD+ in the process

PDC E1 (pyruvate dehydrogenase)

• prosthetic group/coenzyme: TPP (thiamine pyrophosphate)

• Oxidative decarboxylation of pyruvate

  • (Carbanion of TPP) + Pyruvate + NAD⁺ —> Acetyl CoA + NADH + CO2 + (hydroxyethyl-TPP)

• Rate-limiting step

PDC E2 (dihydrolipoyl transacetylase)

• prosthetic group: lipoamide

  • Lipoic acid on a lysine side chain; has a long, flexible, super floppy chain which allows E2 to reach over to E1 and E3 for redox reactions

  • Reactive disulfide bond of the lipoamide can get reduced and must be oxidized again before the reaction can repeat

• Transfer of acetyl group to CoA

  • Hydroxyethyl-TPP (ionized form) + lipoamide —> carbanion of TPP (restored E1) + acetyllipoamide

    • Oxidized lipoamide

    • Moving acetyl from E1 to E2

  • Coenzyme A + acetyllipoamide —> acetyl coA + dihydrolipoamide (reduced)

    • Transfer of 2C from E2 to coA; releases acetyl coA to the mitochondrial matrix

    • Reduced dihydrolipoamide cannot participate in another reaction until it is oxidized again

PDC E3 (dihydrolipoyl dehydrogenase)

• Prosthetic group: FAD

• Regeneration of the oxidized form of lipoamide (E2)

  • Dihydrolipoamide (E2) + FAD —> lipoamide + FADH2

• FADH2 + NAD+ —> FAD + NADH + H+ (rips off the electrons and immediately puts them on NAD+ ultimately)

Citrate synthase

• allosterically inhibited by ATP

• Oxaloacetate binds first (order matters) —> induced fit —> acetyl CoA binds

Aconitase

• citrate <=> isocitrate

• Occurs via dehydration and hydration (taking water off and adding it back on)

• Is a non-heme iron protein; contains an Fe-S cluster (also generally alternates oxidized and reduced state as electrons bind and move through the ETC)

Isocitrate dehydrogenase

• inhibited by high ATP and NADH

• Stimulated by ADP

• Oxidation —> decarboxylation

α-ketoglutarate dehydrogenase complex

• inhibited by ATP and NADH; also succinyl CoA (direct product inhibition)

• Heavily regulated

• Oxidation —> decarboxylation

• At this point, we’ve extracted most of the potential energy; following reactions are just regeneration of oxaloacetate

Succinyl CoA synthetase

• named for the reverse reaction

• Only step in CAC that yields a high energy phosphate bond directly (substrate level phosphorylation)

• Has a histidine residue, which easily reacts to accept and donate the phosphate because its pKa is close to physiological pH (?)

Succinate dehydrogenase

• Fe-S protein embedded in the IMM (only CAC enzyme that’s not soluble in the matrix)

• Is the same as the complex II of the ETC (reduces ubiquionone/coenzyme Q)

CAC total yield (per pyruvate)

• 2 CO2

• 3 NADH

• 1 FADH2

• 1 ATP (GTP)

• Remember, this only runs if there is sufficient O2 (aerobic, but does not use O2 directly) because FAD and NAD+ must be regenerated

Beriberi

• deficiency of thiamine —> required for TPP coenzyme —> can’t make enough α-ketoglutarate dehydrogenase/pyruvate dehydrogenase complex

Metabolic poisons

• poisons such as arsenic and mercury mess up the pyruvate oxidation reactions —> can’t make as much ATP

• Not enough ATP —> tons of CNS effects because it uses a ton of glucose/ATP for things like Na+/K+ pumps, generating and transmitting signals, and the brain’s only fuel is glucose except in extreme starvation

Other pathways

• many of the CAC intermediates also participate in other pathways (Cori cycle, fatty acid/sterol synthesis, glutamate and other amino acid synthesis, etc)

• If you exit, the CAC stops—how do you replenish intermeidates? —> generation of oxaloacetate from pyruvate, as in gluconeogenesis