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Spec Mapping — OCR H420 Module 5.2.2 — Respiration, content statements covering glycolysis as the first stage of respiration, the phosphorylation, lysis, oxidation and substrate-level phosphorylation phases, the net yield per glucose (2 ATP, 2 NADH, 2 pyruvate), and the cytoplasmic location independent of oxygen availability (refer to the official OCR H420 specification document for exact wording).
Glycolysis is the first stage of respiration — the cytoplasmic pathway that splits a single molecule of glucose into two molecules of pyruvate. OCR specification module 5.2.2 requires you to understand the phases of glycolysis, the enzymes, the intermediates, the production of ATP and reduced NAD, and the fate of pyruvate. Glycolysis is universal: every living cell from bacteria to humans uses it, and it is the only ATP-generating pathway that does not require oxygen. Understanding glycolysis is essential for everything that follows.
The pathway was elucidated through the work of the German biochemists Gustav Embden and Otto Meyerhof in the 1920s — hence the alternative name "Embden–Meyerhof pathway". The pathway was reconstructed piece by piece using cell-free yeast extracts (paraphrasing Eduard Buchner's 1897 Nobel-cited demonstration that fermentation could occur without living cells) and step-specific inhibitors. The universality of glycolysis — present in nearly every living organism examined, with the same 10 reactions and the same intermediates — is a strong argument that the pathway evolved before the divergence of bacteria, archaea and eukaryotes, in an early anaerobic world. Paraphrasing the modern evolutionary view, glycolysis is one of the few biochemical pathways whose deep antiquity is unambiguous; it predates oxygen, mitochondria, and the eukaryotic cell itself.
Key Definitions:
- Glycolysis — the splitting of glucose into two molecules of pyruvate, taking place in the cytoplasm, with the net production of 2 ATP and 2 reduced NAD.
- Pyruvate — the 3-carbon product of glycolysis; fate depends on oxygen availability.
- Phosphorylation — the addition of a phosphate group to a molecule, usually using ATP.
- Substrate-level phosphorylation — the direct transfer of a phosphate group from a phosphorylated substrate to ADP, forming ATP (without using the ETC).
- Reduced NAD — NAD that has accepted electrons and a proton (2H) during glycolysis.
Learning objectives — by the end of this lesson you should be able to:
- Describe the four phases of glycolysis (phosphorylation, lysis, oxidation, ATP formation) and state where each occurs.
- Account for the net yield of 2 ATP, 2 reduced NAD and 2 pyruvate per glucose, distinguishing gross from net ATP.
- Explain substrate-level phosphorylation and contrast it with chemiosmotic ATP synthesis.
- Explain how the fate of pyruvate and of reduced NAD differs under aerobic and anaerobic conditions.
- Explain why glycolytic reduced NAD must be shuttled into the mitochondrion, and the consequence for ATP yield.
Glycolysis produces its 2 reduced NAD in the cytoplasm, but the electron transport chain that must re-oxidise them is on the inner mitochondrial membrane. Here lies a problem that older textbooks glossed over and that explains the modern "30–32 ATP" figure: the inner mitochondrial membrane is impermeable to NADH. Reduced NAD cannot simply diffuse into the matrix to reach Complex I. Instead, its electrons (not the molecule itself) must be ferried across by one of two shuttle systems. Which shuttle a cell uses determines how much ATP each cytoplasmic NADH ultimately yields.
This is the more efficient shuttle. In the cytoplasm, oxaloacetate is reduced by NADH to malate (regenerating cytoplasmic NAD⁺); malate crosses the inner membrane via a specific carrier; inside the matrix it is re-oxidised to oxaloacetate, reducing matrix NAD⁺ to NADH. That matrix NADH then feeds Complex I directly. Because the electrons end up on a matrix NADH, each cytoplasmic NADH yields the full ~2.5 ATP. (Oxaloacetate cannot itself cross the membrane, so it returns as aspartate — hence the shuttle's name.) This shuttle is used in tissues where ATP yield matters most.
This is the faster but less efficient shuttle. Cytoplasmic NADH reduces dihydroxyacetone phosphate to glycerol-3-phosphate; a membrane-bound enzyme on the outer face of the inner membrane then re-oxidises it, passing the electrons to FAD, not NAD⁺. Because the electrons now enter the chain via an FADH₂-equivalent (at the ubiquinone level, bypassing Complex I), each cytoplasmic NADH yields only about ~1.5 ATP. Fast-twitch muscle uses this route because it prioritises rapid flux over maximal yield.
| Shuttle | Electrons enter at | ATP per cytoplasmic NADH | ATP from glycolytic NADH per glucose |
|---|---|---|---|
| Malate–aspartate | Complex I (via matrix NADH) | ~2.5 | ~5 |
| Glycerol-3-phosphate | Ubiquinone (via FAD) | ~1.5 | ~3 |
This is precisely why the total ATP yield per glucose is often quoted as a range (30–32) rather than a single number: it depends on which shuttle the tissue uses. The classical "38 ATP" figure ignored the shuttle cost entirely by assuming cytoplasmic NADH could reach Complex I for free. Being able to explain the shuttle mechanism is a strong AO3 discriminator, because it shows you understand that the impermeability of the inner membrane — the same property that makes chemiosmosis possible — also imposes a cost on importing cytoplasmic reducing power.
Glycolysis takes a single 6-carbon glucose molecule and ends up with two 3-carbon pyruvate molecules, plus a small net gain of ATP and reduced NAD. It happens entirely in the cytoplasm (not inside a mitochondrion), does not require oxygen, and does not release any CO₂.
flowchart TB
G[Glucose - 6C] -->|ATP used| G6P[Glucose 6-phosphate - 6C]
G6P --> F6P[Fructose 6-phosphate - 6C]
F6P -->|ATP used| F1P[Fructose 1 6-bisphosphate - 6C]
F1P -->|Lysis| TP2[2 x Triose phosphate - 3C]
TP2 -->|Oxidation + NAD reduced| BP[2 x 1 3-bisphosphoglycerate]
BP -->|ADP phosphorylated| ATP1[2 ATP made]
BP --> GP[2 x Glycerate 3-phosphate]
GP --> PP[2 x Phosphoenolpyruvate]
PP -->|ADP phosphorylated| ATP2[2 more ATP made]
PP --> PYR[2 x Pyruvate - 3C]
OCR simplifies glycolysis into four broad phases. The full biochemistry has 10 steps, but at A-Level you only need the structural outline.
This is sometimes called the "investment phase" — the cell spends ATP before it makes any.
| Item | Per 1 glucose | Per 2 TPs |
|---|---|---|
| ATP used (investment) | 2 | — |
| ATP made (phases 3 + 4) | — | 4 |
| NAD reduced | — | 2 |
| Pyruvate formed | — | 2 |
Although only 2 ATP are made directly, the 2 reduced NADs carry a much larger quantity of energy that will be released later in oxidative phosphorylation (each reduced NAD yields ~2.5 ATP, so an additional ~5 ATP if oxygen is available).
What happens next depends on whether oxygen is available:
| Condition | Fate of pyruvate |
|---|---|
| Aerobic (O₂ present) | Pyruvate is actively transported into the mitochondrial matrix, where it enters the link reaction and then the Krebs cycle. |
| Anaerobic in mammals | Pyruvate is reduced to lactate by lactate dehydrogenase. NAD is regenerated. |
| Anaerobic in plants and yeast | Pyruvate is decarboxylated and reduced to ethanol + CO₂. NAD is regenerated. |
Anaerobic respiration is covered in detail in lesson 11. The key point for now is: in anaerobic conditions, pyruvate must be disposed of in a way that regenerates NAD from reduced NAD, so that glycolysis can continue.
This is one of OCR's favourite questions. The reduced NAD made in glycolysis has to pass its hydrogen (and therefore its electrons) to something eventually — otherwise NAD would all become reduced and glycolysis would stop.
This regeneration of NAD is the whole reason for "fermentation" — without it, glycolysis would stop for lack of NAD, and the cell would make no ATP at all.
OCR does not require detailed knowledge of every enzyme, but you should recognise these key ones for context:
| Enzyme | Reaction |
|---|---|
| Hexokinase | Glucose → glucose 6-phosphate (uses ATP) |
| Phosphofructokinase (PFK) | F6P → F1,6-BP (uses ATP); the main rate-limiting step |
| Aldolase | Splits F1,6-BP into 2 TPs |
| Triose phosphate dehydrogenase | Oxidises TP; reduces NAD; adds phosphate |
| Pyruvate kinase | PEP → pyruvate; makes ATP |
PFK is allosterically regulated by ATP — when the cell has plenty of ATP, PFK activity is inhibited, slowing glycolysis. This is a classic negative feedback loop.
OCR often asks "Explain why glycolysis can continue in the absence of oxygen but oxidative phosphorylation cannot." The answer must include two key steps: (1) glycolysis only requires NAD to be regenerated from reduced NAD, which can be done by reducing pyruvate to lactate (or ethanol); (2) oxidative phosphorylation requires oxygen as the final electron acceptor in the electron transport chain, and without oxygen, electrons back up and the whole chain stops. Missing either point loses marks.
flowchart TB
G[Glucose 6C] -->|Hexokinase + ATP| G6P[Glucose 6-P]
G6P -->|Phosphoglucose isomerase| F6P[Fructose 6-P]
F6P -->|PFK + ATP -- rate-limiting| F16BP[Fructose 1 6-bisphosphate 6C]
F16BP -->|Aldolase -- LYSIS| TP1[Triose-P 3C]
F16BP -->|Aldolase -- LYSIS| TP2[Triose-P 3C]
TP1 -->|G3PDH NAD reduced + Pi| BP1[1 3-bisphosphoglycerate]
TP2 -->|G3PDH NAD reduced + Pi| BP2[1 3-bisphosphoglycerate]
BP1 -->|Phosphoglycerate kinase + ADP -> ATP| GP1[3-phosphoglycerate]
BP2 -->|Phosphoglycerate kinase + ADP -> ATP| GP2[3-phosphoglycerate]
GP1 -->|Mutase + enolase| PEP1[Phosphoenolpyruvate]
GP2 -->|Mutase + enolase| PEP2[Phosphoenolpyruvate]
PEP1 -->|Pyruvate kinase + ADP -> ATP| PYR1[Pyruvate 3C]
PEP2 -->|Pyruvate kinase + ADP -> ATP| PYR2[Pyruvate 3C]
The 10-step pathway can be partitioned into two phases:
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