Glycolysis

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.

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Overview

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]

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The Four Phases

OCR simplifies glycolysis into four broad phases. The full biochemistry has 10 steps, but at A-Level you only need the structural outline.

Phase 1: Phosphorylation

• Glucose is phosphorylated first to glucose 6-phosphate (using 1 ATP) and then isomerised to fructose 6-phosphate.
• Fructose 6-phosphate is phosphorylated again to form fructose 1,6-bisphosphate (using another 1 ATP).
• Cost so far: 2 ATP.
• Phosphorylation makes the molecule more reactive and "locks" it in the cytoplasm (phosphorylated sugars cannot cross the cell membrane).

This is sometimes called the "investment phase" — the cell spends ATP before it makes any.

Phase 2: Lysis (Splitting)

• Fructose 1,6-bisphosphate is split by the enzyme aldolase into two molecules of triose phosphate (TP, 3C).
• This is the "lysis" that gives glycolysis its name (glyco-lysis = sugar-splitting).
• From this point, everything happens twice — once for each TP — so all counts below must be doubled.

Phase 3: Oxidation and Dehydrogenation

• Each TP is oxidised: a hydrogen (H⁺ and electron) is removed and transferred to NAD, forming reduced NAD.
• An inorganic phosphate is also added, forming 1,3-bisphosphoglycerate.
• The phosphate is transferred to ADP, producing ATP by substrate-level phosphorylation.
• Each TP is converted to 3-phosphoglycerate (GP, 3C).
• Per molecule of TP: 1 reduced NAD + 1 ATP. Per glucose: 2 reduced NAD + 2 ATP so far.

Phase 4: ATP Formation

• Each 3-phosphoglycerate is rearranged through a series of intermediates to phosphoenolpyruvate (PEP, 3C).
• A final phosphate is transferred from PEP to ADP, producing another ATP by substrate-level phosphorylation.
• Each PEP becomes pyruvate (3C).
• Per molecule of PEP: 1 ATP. Per glucose: 2 more ATP.

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Net Accounting Per Glucose

Item	Per 1 glucose	Per 2 TPs
ATP used (investment)	2	—
ATP made (phases 3 + 4)	—	4
NAD reduced	—	2
Pyruvate formed	—	2

• Net ATP gain = 4 made − 2 invested = 2 ATP.
• Net reduced NAD = 2.
• Products: 2 pyruvate + 2 ATP + 2 reduced NAD.

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).

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Fate of Pyruvate

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.

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What Happens to the Reduced NAD?

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.

• Aerobic conditions: reduced NAD from glycolysis passes its hydrogens to the electron transport chain inside the mitochondrion (via a shuttle, because reduced NAD itself cannot cross the inner mitochondrial membrane).
• Anaerobic conditions: reduced NAD passes its hydrogens directly to pyruvate (or to acetaldehyde in plants/yeast), reducing it to lactate or ethanol, and regenerating NAD.

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.

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Enzymes of Glycolysis

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.

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Why Does Glycolysis Matter?

• Universal: found in every living organism, from bacteria to plants to animals — strong evidence that it evolved very early in the history of life, before oxygen was abundant in the atmosphere.
• Anaerobic-capable: can happen without oxygen, making it the basis for fermentation-based industries (brewing, baking, yoghurt, cheese).
• Gateway to all later respiration: pyruvate feeds the link reaction and Krebs cycle in aerobic respiration.
• Rapid ATP source: useful in tissues undergoing sudden bursts of activity, like fast-twitch muscle fibres during sprinting.
• Red blood cells depend on it entirely (they have no mitochondria).

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Exam Tip

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.

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Common Exam Mistakes

• Saying glycolysis needs oxygen. It does not — it is anaerobic.
• Writing that glycolysis happens in mitochondria. It happens in the cytoplasm.
• Forgetting to double things after the lysis step. Glucose splits into 2 TPs, so all subsequent yields are doubled.
• Confusing net vs gross ATP. Gross = 4 made. Net = 2 (because 2 were invested).
• Saying pyruvate is the 2-carbon product. Pyruvate is 3-carbon. Acetyl CoA (after the link reaction) is 2-carbon.
• Claiming reduced NAD is made in Krebs only. Reduced NAD is first made in glycolysis, and is also made in the link reaction, Krebs, and β-oxidation.
• Writing "NADH₂" instead of "reduced NAD". OCR uses "reduced NAD".
• Forgetting the role of phosphorylation in making glucose "trapped" in the cytoplasm. The phosphate groups prevent diffusion across the membrane.

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Glycolysis 10-Step Pathway Mermaid

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:

• Investment phase (steps 1–5): glucose phosphorylated twice (2 ATP invested) and split into 2 triose phosphates.
• Payoff phase (steps 6–10): each triose phosphate is oxidised (NAD → NADH), and substrate-level phosphorylation generates 2 ATP per triose phosphate (4 ATP total per glucose).

Net per glucose: $4\,\text{ATP}_{\text{made}} - 2\,\text{ATP}_{\text{invested}} = 2\,\text{ATP net}$4ATPmade​−2ATPinvested​=2ATP net, plus 2 NADH and 2 pyruvate, with no CO₂ produced and no O₂ consumed.

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PFK Regulation — Allosteric Control SVG