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This lesson is mapped to AQA 7402 Section 3.5.1 — Aerobic respiration: glycolysis (refer to the official AQA specification document for exact wording). Glycolysis is the first stage of both aerobic and anaerobic respiration. It is the most ancient, the most ubiquitous, and the only stage of respiration that occurs in every living cell — from anaerobic bacteria living in sulphurous mud, through yeast cells in a brewer's vat, to a human red blood cell that has shed its mitochondria. The pathway takes place in the cytoplasm and does not require oxygen. The word "glycolysis" derives from Greek (γλυκύς, glykys = "sweet"; λύσις, lysis = "splitting"). One molecule of glucose (a 6-carbon hexose) is partially oxidised to two molecules of pyruvate (a 3-carbon α-keto acid), with a small net yield of ATP and the reduced coenzyme NADH.
The pathway is sometimes called the Embden–Meyerhof–Parnas (EMP) pathway in recognition of the German and Polish biochemists who elucidated it between the 1920s and 1940s. Its evolutionary antiquity is striking: every step of glycolysis is conserved across the three domains of life, suggesting that glycolysis already existed in the last universal common ancestor (LUCA), well before the rise of oxygenic photosynthesis ~2.4 billion years ago.
Key Definition: Glycolysis is the metabolic pathway in which one molecule of glucose (6C) is converted into two molecules of pyruvate (3C) in the cytoplasm, yielding a net gain of 2 ATP and 2 reduced NAD (NADH + H⁺) per glucose.
Glycolysis occurs in the cytoplasm for several converging reasons:
Glycolysis can be divided into two phases of approximately equal length: the energy investment phase (steps 1–3) and the energy payoff phase (steps 4–10). At AQA A-Level you are not required to memorise every enzyme name or every intermediate, but you should be able to describe the four key transformations: phosphorylation, lysis (splitting), oxidation (with NAD⁺ reduction), and substrate-level phosphorylation.
Step 1 — Phosphorylation of glucose. Glucose (6C) is phosphorylated to glucose-6-phosphate (6C) using one molecule of ATP. The reaction is catalysed by hexokinase (or glucokinase in liver and pancreas). This investment of one ATP traps glucose inside the cell (the negatively charged phosphate prevents the molecule from leaving via GLUT transporters) and activates it for subsequent reactions.
Step 2 — Isomerisation. Glucose-6-phosphate is rearranged to fructose-6-phosphate (6C) by phosphoglucose isomerase. This converts an aldose sugar into a ketose, positioning the carbonyl group between two hydroxyl-bearing carbons in preparation for splitting.
Step 3 — Second phosphorylation. Fructose-6-phosphate is phosphorylated by a second ATP to form fructose-1,6-bisphosphate (sometimes called hexose bisphosphate, 6C). This is catalysed by phosphofructokinase-1 (PFK-1), the principal regulatory enzyme of the entire pathway. The reaction is strongly exergonic and effectively irreversible under cellular conditions, so the cell uses it as a major control point.
Exam Tip: Two molecules of ATP are invested in steps 1 and 3. This "primes" the glucose molecule, lowering the activation energy of step 4 (the splitting step) so that thermodynamically unfavourable bond rearrangement becomes favourable.
Step 4 — Splitting of hexose bisphosphate. Fructose-1,6-bisphosphate (6C) is cleaved by aldolase into two interconvertible triose phosphates: glyceraldehyde-3-phosphate (G3P or GALP) and dihydroxyacetone phosphate (DHAP). Triose phosphate isomerase rapidly equilibrates the two, so for accounting purposes, one F1,6BP gives two G3P (3C).
Step 5 — Oxidation of triose phosphate. Each G3P is oxidised by glyceraldehyde-3-phosphate dehydrogenase (often written triose phosphate dehydrogenase at A-Level). Two electrons and one proton are transferred to NAD⁺, reducing it to NADH + H⁺ (a second proton enters solution). Simultaneously, an inorganic phosphate (Pi) is added to the substrate to form 1,3-bisphosphoglycerate. This is the critical redox step of the pathway: glucose donates its first electrons, and the cell pays for that later by passing those electrons through the ETC.
Step 6 — Substrate-level phosphorylation (first SLP). A phosphate group is transferred from 1,3-bisphosphoglycerate directly to ADP, forming ATP and 3-phosphoglycerate (3C). Catalysed by phosphoglycerate kinase. With two triose phosphates per glucose, this step produces 2 ATP.
Step 7 — Rearrangement. 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase (positional rearrangement of the phosphate group).
Step 8 — Dehydration. 2-phosphoglycerate is dehydrated by enolase to form phosphoenolpyruvate (PEP), an extraordinarily high-energy intermediate. Loss of water creates a strained, unstable enol-phosphate.
Step 9 — Substrate-level phosphorylation (second SLP). A phosphate group is transferred from PEP to ADP, forming ATP and pyruvate (3C). Catalysed by pyruvate kinase. Again, 2 ATP are produced from two PEP per glucose. The reaction is strongly exergonic and irreversible — the second of the three regulatory checkpoints in glycolysis (the first being hexokinase, the second PFK-1, the third pyruvate kinase).
graph TD
A["Glucose (6C)"] -->|"ATP → ADP<br/>hexokinase"| B["Glucose-6-phosphate"]
B -->|"isomerisation"| C["Fructose-6-phosphate"]
C -->|"ATP → ADP<br/>PFK-1 (regulatory)"| D["Fructose-1,6-bisphosphate"]
D -->|"aldolase"| E["2 × Triose phosphate (3C)"]
E -->|"NAD⁺ → NADH + H⁺<br/>+ Pi"| F["2 × 1,3-bisphosphoglycerate"]
F -->|"→ 2 ATP<br/>(SLP)"| G["2 × 3-phosphoglycerate"]
G --> H["2 × 2-phosphoglycerate"]
H -->|"dehydration"| I["2 × phosphoenolpyruvate (PEP)"]
I -->|"→ 2 ATP<br/>(SLP, pyruvate kinase)"| J["2 × Pyruvate (3C)"]
style D fill:#3498db,color:#fff
style F fill:#27ae60,color:#fff
style J fill:#e67e22,color:#fff
For every molecule of glucose entering glycolysis:
| Product | Quantity |
|---|---|
| Pyruvate (3C) | 2 molecules |
| ATP produced (gross) | 4 molecules (by SLP) |
| ATP used | 2 molecules (steps 1 and 3) |
| Net ATP gain | 2 molecules |
| Reduced NAD (NADH + H⁺) | 2 molecules |
| H₂O released | 2 (from step 8 dehydration) |
| CO₂ released | 0 |
Key Point: The net yield of glycolysis is 2 ATP and 2 NADH per glucose. The 2 NADH molecules carry electrons to the inner mitochondrial membrane (if oxygen is present), where each NADH supports the synthesis of approximately 2.5 further ATP via oxidative phosphorylation — so the indirect yield from cytosolic NADH is significant.
Misconception to avoid: Glycolysis does NOT release any CO₂. Carbon dioxide release begins at the link reaction and continues through the Krebs cycle. A surprisingly common A-Level error is to write "glucose is broken down to CO₂ and water in glycolysis"; this conflates the overall equation of respiration with the first stage only.
Key Definition: Substrate-level phosphorylation (SLP) is the direct transfer of a phosphate group from a phosphorylated substrate molecule to ADP, forming ATP. It does not require oxygen or the electron transport chain.
This contrasts with oxidative phosphorylation, which uses the energy from electron transport to drive ATP synthesis via chemiosmosis (Mitchell's hypothesis, paraphrased in lesson 3). In glycolysis, SLP occurs at two steps:
The fact that SLP occurs without oxygen is biologically critical: cells under hypoxia (low oxygen) or anoxia (zero oxygen) — including red blood cells, fast-twitch muscle fibres during sprinting, and tumour cells in poorly vascularised regions — depend entirely on glycolytic SLP for ATP.
The pathway is regulated at three irreversible steps, with phosphofructokinase-1 (PFK-1) the master control point:
Hexokinase is also feedback-inhibited by its product glucose-6-phosphate, preventing the pointless trapping of glucose if downstream steps are saturated. Pyruvate kinase is activated by fructose-1,6-bisphosphate (feed-forward activation).
Going further: Many cancer cells exhibit the "Warburg effect" — a marked up-regulation of glycolysis even in the presence of oxygen, with lactate as the major product. This is exploited clinically in PET-CT scans, where ¹⁸F-fluorodeoxyglucose lights up rapidly glycolysing tumour tissue.
After glycolysis, pyruvate has three possible fates depending on oxygen availability and cell type:
Exam Tip: The key purpose of fermentation pathways is to regenerate NAD⁺, NOT to produce energy directly. The ATP yield of fermentation is purely glycolytic; the fermentation step itself produces no ATP.
This lesson connects to:
Specimen question modelled on the AQA paper format (6 marks): Describe the role of NAD⁺ in glycolysis and explain why NAD⁺ must be regenerated for glycolysis to continue. (6 marks)
AO breakdown: AO1 3 marks (knowledge of NAD⁺ function and regeneration); AO2 3 marks (application — explaining the consequence of NAD⁺ depletion).
Grade C model answer (~130 words):
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