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10 Steps of Glycolysis: Enzymes, Diagram & ATP Yield

Metabolism · Biochemistry Guide

10 Steps of Glycolysis: Enzymes, Diagram & ATP Yield

The complete ten-step pathway that converts one glucose molecule into two pyruvate — with every enzyme, substrate and energy change, plus the validated assay kits to measure it in your own samples.

Browse Glycolysis Assay Kits →
10ENZYMATIC STEPS
+2NET ATP / GLUCOSE
2NADH PRODUCED
2PYRUVATE MOLECULES

What is glycolysis?

Glycolysis is the first stage of cellular respiration and the primary source of ATP under anaerobic conditions.

Glycolysis is the metabolic pathway that converts one molecule of glucose into two molecules of pyruvate, generating 2 ATP and 2 NADH in the process. This ten-step enzymatic process occurs in the cytoplasm of nearly all living cells and provides both energy and biosynthetic precursors for cellular function. Its speed makes it essential during high energy demand or low-oxygen conditions, where each glucose molecule is broken down progressively and energy is captured in high-energy phosphate bonds.

The overall equation for glycolysis

C6H12O6 + 2 NAD+ + 2 ADP + 2 Pi → 2 Pyruvate + 2 NADH + 2 H+ + 2 ATP

Two ATP are consumed in the preparatory phase and four are generated in the payoff phase — a net gain of 2 ATP per glucose.

Measure glycolysis in your lab

Assay Genie supplies validated, ready-to-use kits to quantify every key output of the glycolytic pathway.

Glycolysis Assay Kit

Glycolysis Assay Kit

A dedicated kit to measure glycolytic flux directly.

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Glucose Assay Kit (Colorimetric)

Glucose Assay Kit

Quantify glucose consumption and uptake in cells and media.

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ATP Colorimetric Assay Kit

ATP Assay Kit

Measure ATP — the primary energy output of glycolysis.

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L-Lactate Assay Kit (Colorimetric)

L-Lactate Assay Kit

Track lactate production under anaerobic and Warburg conditions.

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LDH / Lactate Dehydrogenase Assay Kit

LDH Assay Kit

Assay lactate dehydrogenase activity and cytotoxicity.

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Glucose Uptake Assay Kit (Fluorometric)

Glucose Uptake Assay Kit

Measure cellular glucose uptake in live-cell assays.

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What are the 10 steps of glycolysis?

Glycolysis divides into an energy-investment phase (steps 1–3), preparatory steps (4–5) and a payoff phase (steps 6–10).

The ten reactions of glycolysis
StepEnzymeSubstrate → ProductEnergy change
1HexokinaseGlucose → Glucose-6-phosphate−1 ATP consumed
2Phosphoglucose isomeraseGlucose-6-phosphate → Fructose-6-phosphateNo change
3Phosphofructokinase-1 (PFK-1)Fructose-6-phosphate → Fructose-1,6-bisphosphate−1 ATP (rate-limiting)
4AldolaseFructose-1,6-bisphosphate → DHAP + G3PNo change
5Triose phosphate isomeraseDHAP → Glyceraldehyde-3-phosphateNo change
6G3P dehydrogenaseG3P + NAD+ → 1,3-BPG + NADH+2 NADH produced
7Phosphoglycerate kinase1,3-BPG → 3-Phosphoglycerate+2 ATP generated
8Phosphoglycerate mutase3-PG → 2-PhosphoglycerateNo change
9Enolase2-PG → Phosphoenolpyruvate (PEP)No change
10Pyruvate kinasePEP → Pyruvate+2 ATP generated

The doubling effect occurs because step 4 creates two 3-carbon molecules, so all subsequent reactions run twice per glucose. Net yield: 2 ATP, 2 NADH, 2 pyruvate.

The two phases of glycolysis

1

Energy investment

Steps 1–3 consume 2 ATP to phosphorylate glucose and commit it to the pathway via PFK-1.

2

Cleavage

Steps 4–5 split fructose-1,6-bisphosphate into two interconvertible 3-carbon sugars (G3P).

3

Energy payoff

Steps 6–10 generate 4 ATP and 2 NADH, producing two pyruvate — a net gain of 2 ATP.

How is glycolysis regulated?

Phosphofructokinase-1 (PFK-1, step 3) is the primary rate-limiting enzyme and main control point.

  • Inhibitors: ATP and citrate (signals of sufficient energy) decrease PFK-1 activity.
  • Activators: AMP and ADP (signals of low energy) increase PFK-1 activity.
  • Key activator: Fructose-2,6-bisphosphate, produced by PFK-2, strongly stimulates flux.
  • Hormonal control: Insulin promotes glycolysis; glucagon inhibits it and favours gluconeogenesis.

Measuring pathway output — ATP, lactate and glucose uptake — lets you quantify how these regulatory signals shift glycolytic activity in your model.

Aerobic vs anaerobic glycolysis

The pathway is identical in both, but the fate of pyruvate and how NAD+ is regenerated differ.

Aerobic vs anaerobic glycolysis compared
FeatureAerobicAnaerobic
Oxygen requirementNeeded for NAD+ regeneration via ETCNot required; NAD+ regenerated via lactate
End productPyruvate enters TCA cycle, fully oxidisedPyruvate → lactate (Cori cycle)
ATP yield~30–32 ATP (complete oxidation)2 ATP (glycolysis only)
LocationCytoplasm + mitochondriaCytoplasm only
Clinical relevancePrimary ATP source in resting cellsIntense exercise, hypoxia, cancer (Warburg effect)

Lactate formation via lactate dehydrogenase (LDH) regenerates NAD+ anaerobically, allowing glycolysis to continue.

How glycolysis connects to other pathways

  • TCA cycle: pyruvate → acetyl-CoA for complete oxidation and maximum ATP.
  • Gluconeogenesis: seven of ten steps are reversible, running the pathway backwards to make glucose.
  • Pentose phosphate pathway: glucose-6-phosphate diverted for NADPH and ribose-5-phosphate.
  • Glycogenesis / glycogenolysis: glucose-6-phosphate links to glycogen storage and breakdown.
  • Fatty acid & amino acid pathways: pyruvate and intermediates supply carbon skeletons for biosynthesis.

Studying cellular metabolism?

From glucose uptake to ATP and lactate output, Assay Genie's metabolism assay kits give you validated, publication-ready data — with expert technical support from our PhD team.

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Frequently asked questions

What is the net ATP yield of glycolysis?

The net yield is 2 ATP per glucose. Glycolysis consumes 2 ATP in the preparatory phase (steps 1 and 3) and produces 4 ATP in the payoff phase (steps 7 and 10, each occurring twice per glucose), for a net gain of 2 ATP.

Where does glycolysis occur in the cell?

Glycolysis occurs in the cytoplasm of all cells. Unlike the TCA cycle and electron transport chain (in the mitochondria), glycolytic enzymes are distributed throughout the cytoplasm.

What is the rate-limiting enzyme of glycolysis?

Phosphofructokinase-1 (PFK-1), catalysing step 3, is the primary rate-limiting enzyme. It is inhibited by ATP and citrate and activated by AMP, ADP and fructose-2,6-bisphosphate.

What is the difference between glycolysis and gluconeogenesis?

Glycolysis breaks glucose down into pyruvate and produces ATP; gluconeogenesis synthesises glucose from pyruvate and other precursors, consuming ATP. Seven of the ten reactions are reversible, but three steps must be bypassed by different enzymes.

Why is glycolysis important in cancer cells?

Cancer cells exhibit the Warburg effect, preferentially using anaerobic glycolysis even with oxygen present. This supplies rapid ATP and biosynthetic precursors (nucleotides, amino acids, lipids) to support uncontrolled proliferation.

Related articles

Cellular Respiration: Stages, ATP Production & Pathway GuideRead → Phosphorylation: Types, ATP Synthesis & Cell SignallingRead → Beta Oxidation of Fatty AcidsRead → Cancer Metabolism & the Warburg EffectRead → Spotlight on MitochondriaRead → Amino Acids: Types, Functions & StructureRead →
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Written by Colm Ryan, PhD — Co-founder & CEO of Assay Genie. Colm completed his undergraduate degree in Genetics at Trinity College Dublin, a PhD at the University of Leicester and a post-doc at the IGBMC in Strasbourg, France.

10th Mar 2022 Jahnavi Konduru Naga, Msc

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