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Investment Phase

Investment Phase

4 min read Updated Apr 18, 2026

The first half of glycolysis consumes 2 ATP to prepare glucose for splitting and cleaves the resulting 6-carbon molecule into two 3-carbon molecules.

Glycolysis: glucose to pyruvate in ten steps

Pathway map
Investment · spend 2 ATP Payoff phase everything below happens twice Glucose 1 hexokinase glucokinase in liver and pancreas 1 ATP spent Glucose-6-phosphate phosphoglucose isomerase aldose to ketose Fructose-6-phosphate 2 PFK-1 the committed, rate-limiting step 1 ATP spent Fructose-1,6-bisphosphate aldolase one 6-carbon sugar into two 3-carbon DHAP + glyceraldehyde-3-P triose phosphate isomerase DHAP is converted to G3P 2 × glyceraldehyde-3-P G3P dehydrogenase the only oxidation in the pathway 2 NADH 2 × 1,3-bisphosphoglycerate phosphoglycerate kinase substrate-level phosphorylation 2 ATP 2 × 3-phosphoglycerate phosphoglycerate mutase phosphate moves C3 to C2 2 × 2-phosphoglycerate enolase loses water, stores the energy 2 × phosphoenolpyruvate 3 pyruvate kinase substrate-level phosphorylation 2 ATP 2 × pyruvate Glycogen Pentose phos. NADPH · ribose-5-P Glycerol from fat breakdown: the only part of a triglyceride that can become glucose Lactate LDH · no O₂ Alanine ALT · transamination TO THE MITOCHONDRION PDH → acetyl-CoA → TCA cycle Net per glucose -2 ATP spent +4 ATP made = +2 ATP +2 NADH 2 pyruvate and no O₂ used
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Irreversible (the 3 regulated steps) ATP made NADH made Junction metabolite

Glucose-6-POff-ramps to glycogen synthesis and to the pentose phosphate pathway. This is why hexokinase traps glucose in the cell: G6P carries a charge and cannot leave through GLUT transporters.

DHAPWhere the glycerol backbone of fat joins and leaves. Glycerol from lipolysis enters here, which is the only part of a triglyceride that can become glucose.

PyruvateThe exit into fermentation (lactate), transamination (alanine), or the mitochondrion for PDH and the TCA cycle.

The only three steps you can regulate are the three you cannot reverse. Hexokinase, PFK-1, and pyruvate kinase release far too much free energy to run backwards, so gluconeogenesis has to route around all three. Everything below triose phosphate isomerase happens twice per glucose, which is where the doubled yields come from.

The Five Investment Steps

  1. 1
    Glucose → G6P
    Hexokinase · liver uses glucokinase regulated
    -1 ATP
  2. 2
    G6P → F6P
    Phosphoglucose isomerase · aldose to ketose
    –
  3. 3
    F6P → F1,6BP
    PFK-1 rate-limiting
    -1 ATP
  4. 4
    F1,6BP → DHAP + G3P
    Aldolase · splits 6C into two 3C
    –
  5. 5
    DHAP ⇌ G3P
    Triose phosphate isomerase · both feed the payoff phase
    –

Investment cost: -2 ATP per glucose. End state: 2 G3P, ready for the payoff phase.

Key Points

  • Step 1 (hexokinase): glucose enters the cell via GLUT transporters, then is phosphorylated. Once phosphorylated, glucose cannot leave the cell - it is committed. Hexokinase has a low Km and is in all cells. Liver uses glucokinase (high Km), which only activates at high blood glucose (post-meal).
  • Step 3 (PFK-1): the committed, rate-limiting step. Heavily regulated by ATP (inhibits), AMP (activates), citrate (inhibits), and fructose-2,6-bisphosphate (strongly activates). Hormonal signals (insulin vs. glucagon) control F-2,6-BP levels via PFK-2.
  • Step 4 (aldolase): splits the 6-carbon sugar into two 3-carbon sugars: DHAP and G3P.
  • Step 5 (triose phosphate isomerase): DHAP is converted to G3P. So at the end of step 5, the cell has two G3P molecules, both of which will proceed through the payoff phase.
Why does hexokinase trap glucose in the cell by phosphorylating it?
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Glucose-6-phosphate carries a negative charge and cannot diffuse back through the GLUT transporters that brought glucose in. The phosphate effectively “tags” glucose for retention inside the cell. This is the first committed step in using glucose - once phosphorylated, it must enter glycolysis, glycogen synthesis, or the pentose phosphate pathway.
Why is PFK-1 called the rate-limiting enzyme of glycolysis?
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PFK-1 catalyzes the committed step (fructose-6-P + ATP → fructose-1,6-BP + ADP). This reaction is essentially irreversible and sets the overall rate of glycolysis. PFK-1 is allosterically controlled by many regulators (ATP, citrate inhibit; AMP, fructose-2,6-BP activate), allowing glycolysis to respond to energy state and hormones.
Net energy balance of the investment phase alone (steps 1-5)?
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2 ATP consumed. 0 ATP produced. 1 glucose becomes 2 G3P (glyceraldehyde-3-phosphate). No NADH yet. The investment must be paid back (and exceeded) during the payoff phase for glycolysis to be net energy-producing.
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