PFKFB3 3D Reaction Mechanism Simulator

Loading structural data… (PDB 2AXN · 2DWP · 2I1V · 3QPV · 3QPW)

◉ SLOW-MO · transition-state highlight

References & Databases

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)
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)
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) The C-terminal 440–446 reorganization seen in 3QPU (PPi complex) is not drawn here.
Inhibitors & structures Inhibitor complexes and structure-activity data — J Med Chem 2015;58:3611–25. Boyd S et al. (PMID:25849762)
Sequence & function annotations DNA/gene NCBI Gene 5209 (PFKFB3) · protein UniProt Q16875 · PubChem ATP · RCSB Ligands

Legend & Fidelity

Mechanism fidelity — "associative" is a structure-based proposal, not a direct measurement 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 …". Those hedges are exactly why every arrow here is called an inference — a structure-and-kinetics proposal, not a measurement.
· 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 (His254 Nε2) and acceptor (2-O) line up for an in-line attack on one P. This page's superimposed Michaelis pose measures Nε2 ⋯ P ≈ 4.5 Å (and the nearest Glu323 OE ⋯ 2-O ≈ 3.2 Å); Cavalier et al. 2012 report the donor–acceptor within ≈5.1 Å in their extrapolated pose. In the trapped E~P (3QPV) the transfer has already occurred — His–P is 1.7 Å, His–P⋯O2 3.4 Å — those are not Michaelis distances. His Nε2 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).
· No dissociative / free-metaphosphate (Sₙ1) branch is currently supported 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.
Analysis — what the motion draws vs. what is text-only Rule 30 asks that every narrated sentence be reconstructible from the scene alone. Every claim in the step narrations is carried by one of these:
· Drawn as motion: back-side attack → blue arrow; P–O break / new bond → red/gold dashed bonds; Sₙ2 inversion → the in-flight phosphate is a real equatorial rig whose tick sweeps 180°; PO₃ flight + fadeSwap → product continuity; the in-line O ⋯ P ⋯ O triad (measured 164.5° in the 2DWP coordinates — drawn as a near-collinear line) → collinear dashed legs; covalent phospho-His (E~P) → the 3QPV-derived pose.
· In-scene badges (text allowed — absence claims / non-geometric facts): "no general base in pocket → substrate deprotonates itself (substrate-assisted)" (kinase ③); "associative Sₙ2 · no dissociative free-metaphosphate (SN1) branch currently supported" (kinase ④, pase ②); "TS electronically partially dissociative — hybrid (QM/MM, transferred in; PFKFB3-specific QM/MM not yet published)" (kinase ④).
· Known but not drawable — stated honestly rather than faked: the electronic charge-partition "partially dissociative" is a quantum-mechanical descriptor no geometry can display, so it stays in the badge + narration; "Mg²⁺/Lys169★ (or Arg253·Asn260·His388) stabilize the charge" — the ions/residues are present and ★-marked, but stabilizing electrostatics are an interpretation and framed as such; "(hypothetical, lit.-based)" attaches wherever the papers themselves label a geometry extrapolated. None of these are shortcuts: they are claims that cannot be exhibited by any motion, so they are flagged instead of invented.
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.
Kinetics scope — context, not simulator outputs Stoichiometric context only — the recombinant-human kinase runs K_m(F6P) ≈ 10.2 µM and K_m(ATP) ≈ 16.9 µM — original kinetics: Kim et al. 2007 J Mol Biol 370:14–26 (BRENDA ref 674911) · UniProt Q16875. (A k_cat of 0.4 s⁻¹ sometimes cited for the human kinase could not be traced to either source here.)
Physiological regulation — PFKFB3 in context Induction: PFKFB3 (iPFK-2, "inducible") is a potent stimulator of glycolysis that is up-regulated by hypoxic (HIF-1), mitogenic and inflammatory stimuli and sustains the high glycolytic flux of rapidly proliferating cells such as leukemias and solid tumors (BRENDA EC 2.7.1.105 · Q16875). Allosteric hub: its product F-2,6-BP is the most potent allosteric activator of PFK-1, so PFKFB3 sits upstream of the committed step of glycolysis. Post-translational bias: phosphorylation of the C-terminal Ser461 (UniProt numbering; paper's Ser460 — not in the N-terminal kinase domain) by AMPK / PKA biases the bifunctional enzyme toward kinase activity (faster F-2,6-BP synthesis) — drawn here only as the activity-balance annotation, not as a visible motion. Balance: because the kinase dominates (kinase:pase ≈ 700:1 in the basal state; Cavalier et al. 2012, rising toward ≈ 3000:1 after phosphorylation), the animation always favors synthesis; the kinase activity is accordingly drawn as the strongly favored half-reaction.
Sources — bibliography of this page S1 — PDB 2AXN: first crystal structure of human PFKFB3; Kim SG, Manes NP, El-Maghrabi MR, Lee YH, J Biol Chem 2006;281:2939–44, PMID:16316985 (chain A backbone frame; residue map below) S2 — PDB 2DWP · 2I1V · 2DWO: ternary AMPPCP·F6P kinase complexes revealing the direct substrate–substrate interaction (2-OH of F6P attacks γ-P in-line); J Mol Biol 2007;370:14–26, PMID:17499765; in-line triad computed from 2DWP (the measured O ⋯ P ⋯ O angle is quoted on the Mechanism fidelity card; PG⋯O2(F6P) = 3.20 Å, tools/manifests/pfkfb3.json) S3 — PDB 3QPV · 3QPW: trapped phospho-enzyme (PFKFB3-P·F6-P) and AlF₄⁻ transition-state mimic — His254 nucleophile → covalent phosphohistidine intermediate, Glu323 proton donor, Arg·Asn·His TS stabilization; Proteins 2012;80:1143–53, PMID:22275052; superposed Michaelis contacts P1⋯His254 NE2 = 4.55 Å, OE2⋯2-O = 3.20 Å (manifest) S4 — PDB 3QPU: PPi complex; its C-terminal 440–446 reorganization is not drawn on this page. S5 — Boyd S et al., J Med Chem 2015;58:3611–25, PMID:25849762 (inhibitor complexes and structure–activity data) S6 — Kinetics context: Kim 2007 J Mol Biol 370:14–26 — K_m(F6P) and K_m(ATP) values quoted in the EvidenceView body (recombinant human kinase); BRENDA ref 674911; a sometimes-cited kcat value for the human kinase could not be traced to either source here (kinetics note quoted in the EvidenceView body) S7 — UniProt Q16875 (PFKFB3; residue numbering +1 vs. PDB-author) · NCBI Gene 5209 · PubChem ATP · RCSB Ligands FDP S8 — Repo display conventions: Rule 30 (narrated claims reconstructible from the scene) · 6 geomClaims + in-line triad archived in tools/manifests/pfkfb3.json · active-site superposition RMSD ≈ 0.4 Å (2AXN vs 2DWP/2I1V/3QPV, archived in js/data_pfkfb3.js) · associative Sₙ2 drawn throughout, no dissociative Sₙ1 branch

※ 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 (active-site backbone RMSD ≈ 0.4 Å — archived calculation in js/data_pfkfb3.js) and animations interpolate between real crystallographic coordinates. Where no crystalline state exists, the pose is schematic and labeled as such in the narration.

N-terminal kinase domain (1–245) C-terminal phosphatase domain (246–520) ON PMg²⁺