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Overall structure
First crystal structure of human PFKFB3 — J Biol Chem 2006;281:2939–44. Kim SG, Manes NP, El-Maghrabi MR, Lee YH. (PDB 2AXN · PMID:16316985)
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Kinase mechanism
Ternary AMPPCP·F6P complex reveals a direct substrate–substrate interaction: the 2-OH of F6P attacks the γ-phosphate of ATP in-line — J Mol Biol 2007;370:14–26. (PDB 2DWP · 2I1V · 2DWO · PMID:17499765)
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Phosphatase mechanism
Trapped phospho-enzyme intermediate (PFKFB3-P·F6-P) and AlF₄⁻ transition-state mimic: nucleophilic attack by His → covalent phosphohistidine intermediate; Glu as proton donor; Arg·Asn·His transition-state stabilization — Proteins 2012;80:1143–53. (PDB 3QPV · 3QPW · PMID:22275052)
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Inhibitors & structures
Inhibitor complexes and structure-activity data — J Med Chem 2015;58:3611–25. Boyd S et al. (PMID:25849762)
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Sequence & function annotations
DNA/gene NCBI Gene 5209 (PFKFB3) · protein UniProt Q16875 · PubChem ATP · RCSB Ligands
Legend & Fidelity
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Mechanism fidelity — "associative" is a structure-based proposal, not a direct measurement
A transition state can never be observed directly, so every arrow here is a mechanism inferred from crystallography and kinetics. The PFKFB3 papers themselves hedge: "…suggesting a direct … transfer", "allows the γ-phosphate oxygens to … induce an 'associative' mechanism", "the transition state was extrapolated", "the geometry suggests …".
· Kinase (2DWP): the β,γ-bridge O · γ-P · 2-OH of F6P lie on one line; the short 2-OH⋯γ-P distance lets a γ-P oxygen act as the general base → associative, in-line Sₙ2, inversion, single step, no phosphoenzyme — J Mol Biol 2007;370:14–26 (PMID:17499765).
· Phosphatase (3QPV · 3QPW): donor and acceptor within ≈5.1 Å on one line; His Nε attacks the 2-P through a transient bipyramidal phosphorane (pentacoordinate) → associative, in-line, inversion in both half-reactions — Proteins 2012;80:1143–53 (PMID:22275052).
· There is no dissociative / free-metaphosphate (Sₙ1) branch proposed for either PFKFB3 half-reaction. Our animation draws associative Sₙ2 throughout, and each transition state is labeled "(hypothetical, lit.-based)" to match the papers' own hedged framing.
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Why is "Show AlF₄⁻ TS analog" a checkbox?
AlF₄⁻ (aluminum tetrafluoride) is not a substrate — it is a transition-state analog soaked onto PFKFB3 in the crystal (PDB 3QPW). Sitting in the 2-phosphate pocket it reproduces the pentacoordinate, trigonal-bipyramidal geometry of the phosphorane TS: the four F atoms stand where the equatorial P–O bonds would be, and the axial O(s) mark where the incoming nucleophile and leaving group go. Flipping the box overlays that real crystal TS mimic at the histidine site so the observed geometry can be compared with the stylized PO₃ flight. It is a checkbox because AlF₄⁻ is an inhibitory artifact — it is not an on-pathway species — so it stays off by default and appears only when asked.
※ On-screen active-site residue numbers follow the UniProt Q16875 standard numbering, which is +1 relative to
the PDB author numbering used in the structure papers (e.g., catalytic His254 = paper's His253). The protein backbone uses chain A
coordinates of 2AXN; every ligand state was superposed into one common frame (RMSD ≈ 0.4 Å) and animations interpolate between
real crystallographic coordinates. Where no crystalline state exists, the pose is schematic and labeled as such in the narration.