Glycolysis

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(c) 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.

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: