Metabolism — glycolysis (step 10)
Pyruvate kinase: substrate-level phosphorylation
How the Pyruvate kinase works, in 3 steps. Each step shows the structures and the curved arrows that move the electrons.
In short. A 3-step mechanism. Typical reagents: Phosphoenolpyruvate (PEP), ADP, Mg²⁺, K⁺, pyruvate kinase. Conditions: Cytosol, pH ≈ 7.4, 37 °C. Bonds formed: ATP (P–O bond); pyruvate C=O; bonds broken: PEP P–O (enol), enol C=C. Stereochemistry: In-line phosphoryl transfer at P; enol protonated on a defined face. Rate and selectivity: Irreversible; ΔG°′ ≈ −31 kJ/mol; regulated by fructose-1,6-bisphosphate.
At a glance
Step 1: ADP attacks the phosphate of PEP
ADP's terminal oxygen attacks the phosphorus of phosphoenolpyruvate's phosphate group in-line, displacing the enolpyruvate oxygen as the leaving group — the same in-line phosphoryl transfer mechanism as hexokinase's first step, just running in the ATP-generating direction with the nucleophile and leaving group swapped.
Step 2: ATP and enolpyruvate released
This releases the free enol form of pyruvate, which is high in energy specifically because it isn't yet tautomerized to the far more stable ketone.
Step 3: Tautomerization to pyruvate
Rapid, essentially irreversible tautomerization to the keto form of pyruvate is what makes this whole phosphoryl transfer thermodynamically favorable — this large, one-way driving force (enol-to-ketone tautomerization) is exactly why this step, along with the analogous phosphoglycerate kinase step, is where glycolysis directly generates ATP without needing the electron transport chain.
Abbreviations and terms
- ATP
- Adenosine triphosphate. The cell’s energy currency; transfers phosphate groups.
- ADP
- Adenosine diphosphate. ATP after losing one phosphate.
How it compares with related reactions
| Pyruvate kinase | Enolase | GAPDH | |
|---|---|---|---|
| Reagents | Phosphoenolpyruvate (PEP), ADP, Mg²⁺, K⁺, pyruvate kinase | 2-Phosphoglycerate, 2 Mg²⁺, enolase (Lys345 base, Glu211 acid) | Glyceraldehyde-3-phosphate (G3P), NAD⁺, inorganic phosphate (Pi), GAPDH (Cys149, His176) |
| Conditions | Cytosol, pH ≈ 7.4, 37 °C | Cytosol, pH ≈ 7, 37 °C | Aqueous, pH ≈ 7.4, 37 °C (cytosol) |
| Stereochemistry | In-line phosphoryl transfer at P; enol protonated on a defined face | Anti elimination of H and OH; reversible | Hydride delivered to one face of NAD⁺ (pro-R) |
| Rate / selectivity | Irreversible; ΔG°′ ≈ −31 kJ/mol; regulated by fructose-1,6-bisphosphate | ΔG°′ ≈ +1.8 kJ/mol; PEP has a very high phosphoryl-transfer potential | Fast and reversible; couples oxidation to phosphate capture |
Test yourself
Try to answer each question before opening it.
Which reagents and conditions are typical for the Pyruvate kinase?
Phosphoenolpyruvate (PEP), ADP, Mg²⁺, K⁺, pyruvate kinase. Conditions: Cytosol, pH ≈ 7.4, 37 °C.
Which bonds are formed and which are broken?
Formed: ATP (P–O bond); pyruvate C=O. Broken: PEP P–O (enol), enol C=C.
What is the stereochemical outcome?
In-line phosphoryl transfer at P; enol protonated on a defined face
What controls the rate or the selectivity?
Irreversible; ΔG°′ ≈ −31 kJ/mol; regulated by fructose-1,6-bisphosphate
Study guides that help with this reaction
- How to learn reaction mechanisms (without memorising everything)
- Curved-arrow notation: rules and the mistakes students make