Metabolism — glycolysis (step 3)
Phosphofructokinase-1: committed phosphorylation
How the Phosphofructokinase-1 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: Fructose-6-phosphate (F6P), ATP·Mg²⁺, PFK-1 (active-site base, Asp127). Conditions: Aqueous, pH ≈ 7.4, 37 °C, allosterically regulated. Bonds formed: C1–O–P (F-1,6-BP); ADP; bonds broken: O–H (C1); P–O (γ-phosphoanhydride). Stereochemistry: In-line attack at Pγ: inversion at phosphorus. Rate and selectivity: Committed step: inhibited by ATP and citrate, activated by AMP and fructose-2,6-bisphosphate.
At a glance
Step 1: Base activates the C1–OH
Fructose-6-phosphate's C1 hydroxyl is positioned against ATP's terminal (γ) phosphate by the enzyme, with Mg²⁺ coordinating the ATP phosphates. An active-site base deprotonates the OH, activating it as a nucleophile.
Step 2: In-line attack at Pγ
The activated oxygen attacks the γ-phosphorus in-line, opposite the departing ADP, in the same SN2-like phosphoryl transfer used by hexokinase. A pentacoordinate phosphorane-like transition state forms.
Step 3: F-1,6-BP and ADP
The products are fructose-1,6-bisphosphate and ADP. This is the committed, rate-controlling step of glycolysis, which is why PFK-1 is the main regulatory site: ATP and citrate inhibit it, while AMP and fructose-2,6-bisphosphate activate it.
Abbreviations and terms
- R
- R group. Any carbon substituent (alkyl, aryl…) whose identity does not matter for the mechanism.
- TS
- Transition state. The highest-energy point on the pathway between two minima; it cannot be isolated.
- SN2
- Bimolecular nucleophilic substitution. One concerted step with backside attack and inversion; rate = k[RX][Nu].
- ATP
- Adenosine triphosphate. The cell’s energy currency; transfers phosphate groups.
- ADP
- Adenosine diphosphate. ATP after losing one phosphate.
- PFK-1
- Phosphofructokinase-1. The key regulated, committed step of glycolysis.
- Asp
- Aspartate. Acidic amino acid.
How it compares with related reactions
| Phosphofructokinase-1 | Enolase | GAPDH | |
|---|---|---|---|
| Reagents | Fructose-6-phosphate (F6P), ATP·Mg²⁺, PFK-1 (active-site base, Asp127) | 2-Phosphoglycerate, 2 Mg²⁺, enolase (Lys345 base, Glu211 acid) | Glyceraldehyde-3-phosphate (G3P), NAD⁺, inorganic phosphate (Pi), GAPDH (Cys149, His176) |
| Conditions | Aqueous, pH ≈ 7.4, 37 °C, allosterically regulated | Cytosol, pH ≈ 7, 37 °C | Aqueous, pH ≈ 7.4, 37 °C (cytosol) |
| Stereochemistry | In-line attack at Pγ: inversion at phosphorus | Anti elimination of H and OH; reversible | Hydride delivered to one face of NAD⁺ (pro-R) |
| Rate / selectivity | Committed step: inhibited by ATP and citrate, activated by AMP and fructose-2,6-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 Phosphofructokinase-1?
Fructose-6-phosphate (F6P), ATP·Mg²⁺, PFK-1 (active-site base, Asp127). Conditions: Aqueous, pH ≈ 7.4, 37 °C, allosterically regulated.
Which bonds are formed and which are broken?
Formed: C1–O–P (F-1,6-BP); ADP. Broken: O–H (C1); P–O (γ-phosphoanhydride).
What is the stereochemical outcome?
In-line attack at Pγ: inversion at phosphorus
What controls the rate or the selectivity?
Committed step: inhibited by ATP and citrate, activated by AMP and fructose-2,6-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