Glycolysis, Pyruvate Dehydrogenase and the Citric Acid Cycle Explained

Glycolysis

Glycolysis: glucose + 2 ADP + 2 Pi + 2 NAD+ → 2 ATP + 2 pyruvate + 2 NADH + 2 H+

Step 1

Hexokinase phosphorylates glucose, keeping it inside the cell. This is a coupled reaction, making it spontaneous.

Step 2

Isomerisation.

Step 3

ATP coupling makes the non-spontaneous reaction spontaneous (PFK).

Step 4

Lysis.

Step 5

DHAP is converted to G3P by TPI.

Step 6

G3P dehydrogenase catalyses a redox reaction: NAD+ → NADH + H+.

Step 7

Phosphoglycerate kinase catalyses substrate-level phosphorylation of ADP, and the payoff phase repays the ATP debt.

Step 8

The phosphate is moved from C3 to C2 via phosphoglycerate mutase.

Step 9

Enolase dehydrates the substrate to form enol, forming PEP.

Step 10

Pyruvate kinase catalyses second-level phosphorylation of ADP, forming 2 ATP.

Fates of Pyruvate

• Yeast: regenerates NAD+ via pyruvate decarboxylase, acetaldehyde and alcohol dehydrogenase, producing ethanol.
• Animals: pyruvate → lactate via lactate dehydrogenase.

Gluconeogenesis

Gluconeogenesis consumes 4 ATP + 2 GTP + 2 NADH (ΔG = different). It uses these bypass reactions:

• Bypass 1a: Pyruvate carboxylase burns 1 ATP and makes oxaloacetate, a 4C molecule. It uses a biotin cofactor, is located in the mitochondria, and oxaloacetate must be reduced to malate to reach the cytoplasm.
• Bypass 1b: PEP carboxykinase converts oxaloacetate back to a 3C molecule, burning GTP and making PEP.
• Bypass 2: Fructose-1,6-bisphosphatase hydrolyses the phosphate ester, returning to F6P. Reciprocal regulation: citrate up, AMP down, F-2,6-BP down.
• Bypass 3: Glucose-6-phosphatase converts G6P to glucose in the ER. It is present in kidney and liver, not brain or muscle.

Pyruvate Dehydrogenase (PDH) Complex

The PDH complex uses the following cofactors:

• TPP (vitamin B1), non-covalently bound to E1.
• Lipoic acid, covalently bound to lysine on E2, forming lipoamide. Disulfide is reduced to dithiol.
• CoA (vitamin B5), a non-covalent E2 substrate. The thiol of E2 covalently binds an acyl group to form a thioester (acetyl-CoA).
• NAD+ (vitamin B3, niacin), an electron carrier that carries two-electron transfer reactions.
• FAD (vitamin B2, riboflavin), an isoalloxazine ring existing in the 3 oxidation state, serving as an electron carrier in one- or two-electron transfer reactions.

Reaction Steps

1. Decarboxylation and hydroxyethyl-TPP formation: TPP binds to E1 and reacts with pyruvate, generating reactive hydroxyethyl-TPP and CO2.
2. Oxidation/reduction: The hydroxyethyl group is transferred from E1 to the lipoamide arm of E2. Hydroxyethyl-TPP is oxidised, lipoamide is reduced, forming a thioester intermediate.
3. Acetyl-CoA formation: E2 transfers the acetyl group to CoA, producing acetyl-CoA and reduced lipoamide.
4. FAD reduction/regeneration of oxidised lipoamide: The reduced lipoamide swings to the E3 active site. E3 contains covalently linked FAD, which acts as an electron acceptor, re-oxidising the lipoamide of E2.
5. FADH2 oxidation/NAD+ reduction: The disulfide of E3 re-oxidises FADH2 and NAD+ is reduced to NADH. FAD serves as an important intermediate, with the disulfide of E3 oxidised by FAD and FADH2 oxidised by NAD+.

TCA (Citric Acid) Cycle

Reaction 1: Condensation

Hydrolysis of the thioester releases CoA and forms citrate. The COOH groups in citrate are not equivalent.

Step 2: Isomerisation

Citrate is a poor substrate due to its tertiary alcohol. The reaction swaps the tertiary alcohol to a secondary alcohol, which is readily oxidised. This dehydration/hydration reaction is catalysed by aconitase, is stereospecific, removes the pro-R (H) of the pro-R arm, and forms isocitrate.

Step 3: Oxidative Decarboxylation

Isocitrate dehydrogenase abstracts an H2 from isocitrate, passing a pair of electrons to NAD+ to produce NADH and releasing a proton. Oxalosuccinate undergoes β-decarboxylation, in which the carboxyl group attached to the β-carbon relative to the keto group is removed as CO2. IDH requires a cofactor to facilitate the reaction. NADH and ATP are allosteric inhibitors; ADP acts as an allosteric activator. The product is α-ketoglutarate.

Step 4: Second Oxidative Decarboxylation

α-Ketoglutarate is oxidised to succinyl-CoA by α-ketoglutarate dehydrogenase. The energy of decarboxylation is conserved in the high-energy thioester bond of -S-CoA. α-Ketoglutarate dehydrogenase is a multienzyme complex analogous to PDH.

Step 5: Succinyl-CoA Hydrolysis and GTP Synthesis

The unstable high-energy thioester bond is hydrolysed (negative ΔG), coupled with GTP synthesis (positive ΔG) from GDP. Succinyl-CoA is converted to succinate and CoA is released. Energy stored in the thioester bond is conserved in GTP. The reaction is catalysed by succinyl-CoA synthetase.