Citric Acid Cycle Notes

Pyruvate from glycolysis is oxidized to $H2O$ and $CO2$ during respiration, an aerobic catabolic phase.
Respiration (broader sense): Multicellular organism's uptake of $O2$ and release of $CO2$ .
Cellular Respiration (biochemical sense): Molecular processes where cells consume $O2$ and produce $CO2$ .

Stage 1: Oxidation of organic fuels (glucose, fatty acids, amino acids) into two-carbon fragments as acetyl group of acetyl-CoA.
Stage 2: Acetyl groups enter the citric acid cycle and are oxidized to $CO2$ . Energy is conserved via reduced electron carriers NADH and $FADH2$ .
Stage 3: Reduced coenzymes ( $NADH$ and $FADH2$ ) are oxidized, releasing protons ( $H^+$ ) and electrons. Electrons are transferred to $O2$ (final electron acceptor) through the respiratory chain. Energy released is conserved as ATP via oxidative phosphorylation.

Sugars and fatty acids are degraded to acetyl-CoA before entering the citric acid cycle.
Some amino acids are also degraded to cycle intermediates.
Pyruvate, derived from glucose via glycolysis, is oxidized to acetyl-CoA and $CO_2$ by the pyruvate dehydrogenase (PDH) complex.
- Location: Mitochondria (eukaryotes) and cytosol (prokaryotes).
Overall Reaction: Oxidative decarboxylation, which is irreversible. The carboxyl group is removed from pyruvate as $CO_2$ .
The PDH complex contains three enzymes:
- Pyruvate dehydrogenase (E1)
- Dihydrolipoyl transacetylase (E2)
- Dihydrolipoyl dehydrogenase (E3)
It requires five coenzymes:
- Thiamine pyrophosphate (TPP)
- Flavin adenine dinucleotide (FAD)
- Coenzyme A (CoA)
- Nicotinamide adenine dinucleotide (NAD)
- Lipoate

Step 1: Pyruvate reacts with TPP bound to pyruvate dehydrogenase (E1), undergoing decarboxylation to form a hydroxyethyl derivative.
Step 2: E1 transfers two electrons and the acetyl group from TPP to the oxidized lipoyllysyl group of dihydrolipoyl transacetylase (E2), forming acetyl thioester of the reduced lipoyl group.
Step 3: Transesterification: The –SH group of CoA replaces the –SH group of E2, yielding acetyl-CoA and the fully reduced (dithiol) form of the lipoyl group.
Step 4: Dihydrolipoyl dehydrogenase (E3) promotes transfer of two hydrogen atoms from the reduced lipoyl groups of E2 to the FAD prosthetic group of E3, restoring the oxidized form of the lipoyllysyl group of E2.
Step 5: The reduced $FADH_2$ of E3 transfers a hydride ion to $NAD^+$ , forming $NADH$ . The enzyme complex is now ready for another catalytic cycle.

Acetyl-CoA donates its acetyl group to oxaloacetate (four-carbon compound) to form citrate (six-carbon).
Citrate is transformed into isocitrate (six-carbon).
Isocitrate is dehydrogenated with loss of $CO_2$ to yield α-ketoglutarate (five-carbon).
α-Ketoglutarate undergoes loss of a second molecule of $CO_2$ and ultimately yields succinate (four-carbon).
Succinate is enzymatically converted in three steps into oxaloacetate (four-carbon), which can react with another molecule of acetyl-CoA.
In each cycle turn, one acetyl group (two carbons) enters, and two $CO_2$ molecules leave. Oxaloacetate is regenerated, so there's no net removal.
Four of the eight steps are oxidations, conserving energy in the form of reduced coenzymes $NADH$ and $FADH_2$ .

Condensation of acetyl-CoA and oxaloacetate to citrate (Citrate synthase)
Dehydration (and hydration) of citrate to isocitrate (Aconitase)
Oxidative decarboxylation of isocitrate to α-ketoglutarate (Isocitrate dehydrogenase)
Oxidative dehydrogenation of α-ketoglutarate to succinyl-CoA (α-ketoglutarate dehydrogenase complex)
Substrate-level phosphorylation of succinyl-CoA to succinate (Succinyl-CoA synthase)
Dehydrogenation of succinate to fumarate (Succinate dehydrogenase)
Hydration of fumarate to malate (Fumarase)
Dehydrogenation of malate to oxaloacetate (Malate dehydrogenase)

Glycogen metabolism is regulated by insulin, glucagon, epinephrine, and allosteric regulators to avoid wasting energy.
Carbon atom flow from pyruvate into and through the citric acid cycle is tightly regulated at two levels:
- Conversion of pyruvate to acetyl-CoA (PDH complex reaction).
- Entry of acetyl-CoA into the cycle (citrate synthase reaction).
The cycle is also regulated at the isocitrate dehydrogenase and α-ketoglutarate dehydrogenase reactions.

The PDH complex of mammals is inhibited by ATP, acetyl-CoA, and NADH (products of the reaction).
Inhibition is enhanced when long-chain fatty acids are available.
The PDH complex is activated by AMP, CoA, and NAD, which accumulate when too little acetate flows into the cycle.

Three factors govern the flux rate through the cycle:
- Substrate availability
- Product inhibition
- Allosteric feedback inhibition.
Three exergonic steps (citrate synthase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) can become rate-limiting.
Substrate availability for citrate synthase (acetyl-CoA and oxaloacetate) varies with the metabolic state.
High [NADH]/[NAD+] inhibits isocitrate and α-ketoglutarate dehydrogenase reactions.
Product accumulation inhibits all three limiting steps:
- Succinyl-CoA inhibits α-ketoglutarate dehydrogenase and citrate synthase.
- Citrate blocks citrate synthase.
- ATP inhibits citrate synthase and isocitrate dehydrogenase. ADP relieves ATP inhibition of citrate synthase.
In vertebrate muscle, $Ca^{2+}$ activates isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, as well as the PDH complex.
Substrate and intermediate concentrations in the citric acid cycle set the flux through this pathway to maintain optimal ATP and NADH concentrations.