Phosphofructokinase-1 - 3D Mechanism Simulator

Structure (frame) E. coli PFK in complex with the reaction products F1,6BP + ADP + Mg²⁺ 1PFK (Shirakihara Y & Evans PR, J Mol Biol 204:973 1988, PMID:2975709; DOI 10.2210/pdb1PFK/pdb). Active-state scaffold of Geobacillus (Bacillus) stearothermophilus PFK with F6P bound 4PFK (Evans PR, Farrants GW & Hudson PJ, Philos Trans R Soc Lond B 293:53 1981, PMID:6115424). Human PFK-M shares the conserved ASKHA catalytic machinery, but its own structures top out at the dissociated muscle dimer 4OMT (6 Å, 2014); the eukaryotic R/T cryo-EM frames shown (8W2G / 8W2H) are the liver isozyme PFK-L (2.6 Å), referenced by labels, not PFK-M meshes. Residue numbering here follows the geometry frame 4PFK (G. stearothermophilus: Asp127/Arg171) — not the differently numbered E. coli 1PFK (see "Residue numbering" below).
① F6P binding — a real crystal pose, not a schematic Unlike PGI's sugar (which lives in a trap-analog pocket), F6P here is the actual deposited ligand of the 4PFK complex (frame f6p@4PFK). Measured in that frame: Asp127 Oδ2 ⋯ F6P O1 (1-OH) = 3.02 Å and Arg171 NH1 ⋯ O1 = 2.98 Å. The 6-phosphate is cradled by the partner subunit's Arg162′ (chain B — not rendered in this single-frame snapshot; rendering it needs the dimer). The chain-A Arg162 in this frame sits ≈ 30 Å away — it is not the 6-P grip, and the old "Arg162/171/25 network grips the 6-P" phrasing has been dropped (see geomClaims, Rule 34).Source: PDB 4PFK (frame f6p@4PFK) — deposited F6P ligand pose; measured separations: Asp127·OD2 ⋯ F6P·O1 = 3.02 Å (geomClaim pfk-asp-o1) and Arg171·NH1 ⋯ O1 = 2.98 Å (printed reading, carries no declared claim); 6-P cradle = partner-subunit Arg162′ (chain B, not rendered); same-chain Arg162 ≈ 30 Å catalytically irrelevant (geomClaim pfk-arg162-o1)
② Mg·ATP — why the scaffold is schematic No PFK-1·F6P·MgATP Michaelis ternary crystal has ever been captured. The adenosine part is therefore the ADP scaffold of the reaction-product complex (its N-/P-pocket pose is real), riveted to a hand-built γ-PO₃ to make ATP. The γ-P donor seat sits ≈ 3.4 Å from O1 along the β-P line — that number is a runtime assembly seam of our reconstruction, not a crystallographic measurement (it carries no geomClaim; see "Precision vs. accuracy"). Mg²⁺ rides in as Mg·ATP and ion-pairs the phosphate oxygens.Source: no PFK-1·F6P·MgATP Michaelis ternary crystal exists — the adenosine is the deposited ADP scaffold of PDB 1PFK (product complex) + a hand-built γ-PO₃ rivet; γ-P seat ≈ 3.4 Å = runtime assembly seam, not a crystallographic number (species Mg·ATP in tools/manifests/pfk.json; see "Why is the ATP a scaffold + γ-PO₃ schematic?" card)
③ Asp127 deprotonates the 1-OH — mutagenesis-pinned PROPOSAL Asp127 ★ is the general base: its carboxylate sits 3.02 Å from the F6P 1-OH oxygen in the crystal (measured) and removes that proton so 1-O⁻ is the nucleophile. Site-directed mutagenesis pins the identity: Asp127→Ser reduces the forward turnover 18,000× and the reverse 3,100×, and destabilizes the product complex — DOI 10.1038/327437a0 (Hellinga HW & Evans PR, Nature 327:437, 1987, PMID:2953977). Arg171→Ser costs only ≈ 3.4× — so Arg171 (2.98 Å from O1, measured) is drawn as a modest frame, not a strong TS lock. Asp127's deprotonated carboxyl is drawn as the active base; its effective pKa is shifted up toward neutrality by its electrostatic partner Asp129 — Berger SA & Evans PR, Biochemistry DOI 10.1021/bi00153a017 (1992, PMID:1390710). Note the pH-dependence of catalysis tracks a pK ≈ 6 group that Lainé R et al. explicitly assign not to Asp127/Asp129 themselves — Eur J Biochem DOI 10.1111/j.1432-1033.1992.tb17148.x (1992). The gold H⁺ ball and its O1→OD2 path are [schematic] (Rule 23) — the proton hop itself is the inference, marked PROPOSAL.Source: PDB 4PFK — Asp127·OD2 ⋯ F6P·O1 = 3.02 Å measured (geomClaim pfk-asp-o1); mutagenesis pins the base: DOI 10.1038/327437a0 (Hellinga & Evans 1987, Nature 327:437): Asp127→Ser = 18,000×/3,100× losses, Arg171→Ser ≈ 3.4×; Asp129 pKa-partner shift: DOI 10.1021/bi00153a017 (Berger & Evans 1992); pK ≈ 6 group ≠ Asp127/Asp129: DOI 10.1111/j.1432-1033.1992.tb17148.x (Lainé 1992); the proton hop itself = PROPOSAL, drawn [schematic] (Rule 23)
④ γ-P transfer — in-line, associative, inverted (PROPOSAL) The 1-O⁻ attacks the γ-P back-side, in line with the P−O bond being broken — an associative S_N2 that inverts the phosphate (as seen for the related sugar kinases; the inversion was shown by stereochemical assay — Jarvest & Lowe 1981, cited in Shirakihara & Evans 1988). The residue chemistry is therefore PROPOSAL drawing; what the 1PFK crystal does prove is the destination: the product complex's P1 sits 1.89 Å from where the F6P O1 was (measured; geomClaim pfk-p1-o1) — the transferred phosphate lands exactly on the attacking oxygen, so the F6P→F1,6BP crossfade happens in place. (Whether the PFK transition state is strictly associative or leans dissociative-metaphosphate is debated — Hellinga & Evans 1987 caution the distinction blurs in enzymes; the in-line, inverting geometry is common ground, and the associative reading is the textbook default we draw.)Source: product-state footprint measured in PDB 1PFK/4PFK: F1,6BP·P1 ⋯ F6P·O1 = 1.89 Å (geomClaim pfk-p1-o1) — the transferred phosphate lands on the attacking 1-O; in-line inverting S_N2 + stereochemical assay (Jarvest & Lowe 1981, via Shirakihara & Evans 1988) and the associative-vs-dissociative TS debate (Hellinga & Evans 1987) = PROPOSAL, lit.-based — no PFK transition state is observed
⑤ Release + regulation — committed, allosterically gated F1,6BP is handed to aldolase (step 4); this is the committed, essentially irreversible step of glycolysis — the main flux-control site in most tissues. Regulation differs by domain: bacterial PFK (this frame) shows homotropic F6P cooperativity and allosteric ADP/GDP activation · PEP inhibition at a regulatory site (4PFK); eukaryotic PFK is a tetramer whose R↔T switch is controlled by AMP and fructose-2,6-bisphosphate (activate) vs ATP and citrate (inhibit), with a rotated regulation domain (cryo-EM 8W2G/8W2H of the liver isozyme PFK-L — depicted only by labels; see "Static protein"). The activator fructose-2,6-bisphosphate is made by PFKFB3 — a different enzyme, EC 2.7.1.105 (see the PFKFB3 page); BRENDA likewise warns "Not identical with EC 2.7.1.105".Source: committed, essentially irreversible step, F1,6BP handed to aldolase (next enzyme); bacterial homotropic F6P cooperativity + allosteric ADP/GDP activation · PEP inhibition at a regulatory site: PDB 4PFK; eukaryotic R↔T switch (AMP and fructose-2,6-bisphosphate activate · ATP and citrate inhibit, regulation-domain rotation) = cryo-EM 8W2G/8W2H of the liver isozyme PFK-L, label-referenced only (see "Static protein" card); F-2,6-BP maker = PFKFB3 page, EC 2.7.1.105 — "not identical with EC 2.7.1.105" (BRENDA)
Quantitative kinetics (human PFK-M, UniProt P08237) Human muscle PFK-1 (PFK-M): kcat ≈ 8.11 s⁻¹ (F6P), K0.5(F6P) ≈ 147.1 µM (Km ≈ 0.147 mM), kcat/K0.5 ≈ 55 s⁻¹·mM⁻¹ (8.11 s⁻¹ ÷ 0.1471 mM; the paper's printed 3.8×10⁻² is a Vmax/K artifact with mixed units), K0.5(ATP) ≈ 152 µM, Hill h ≈ 1.82 (positive F6P cooperativity) — among the three PFK-1 isozymes PFK-M is the most resistant to ATP inhibition. Sources: DOI 10.1042/bcj20200656 (Fernandes PM et al., Biochem J 477(22):4425–4441 2020, recombinant human PFK-1 isozymes PFK-M/L/P); BRENDA EC 2.7.1.11 (P08237) — which also notes the absolute β-anomer stereospecificity for D-fructofuranose-6-P and the 2.7.1.105 distinction. Numbers are single kinetic values, not a flux model.
Reaction coordinate & protonation states [schematic] One chemical step: deprotonation (Asp127) then a single associative phosphoryl-transfer TS. Peak height is stylistic — no PFK TS is directly measurable. Protonation states at pH 7: the catalysis pH-dependence tracks a pK ≈ 6 group that Lainé 1992 assigns not to Asp127/Asp129 themselves — Asp127's carboxyl is drawn deprotonated as the general base (its effective pKa lifted toward neutrality by the Asp129 partner; Hellinga & Evans 1987 · Berger & Evans 1992). Bookkeeping: the H⁺ of the net equation (F6P + ATP → F1,6BP + ADP + H⁺) is operationally this abstracted 1-OH proton, carried on Asp127 and released at product exit — the real acid–base exchange with the product phosphates and bulk water is simplified. This screen draws an associative TS; whether the true PFK TS is associative or dissociative-metaphosphate is still debated in the field (see ④). Mg²⁺ bridges the γ-P and β-P oxygens, Arg171 provides the steady cation.
Sequence & isozymes DNA/gene NCBI Gene 5213 (PFKM) · protein UniProt P08237. Three human PFK-1 isozymes: PFK-M (muscle; this page's kinetic reference), PFK-L (liver), PFK-P (platelet/brain) — products of different genes; deficiency of PFK-M causes glycogen storage disease type VII (Tarui disease). Residue numbering caveat: the catalysis numbering on this page (Asp127★, Arg171) follows the 4PFK frame (G. stearothermophilus); the E. coli 1PFK sequence numbers the same catalytic residues Asp128/Arg172, and human PFK-M differs again — the conserved ASKHA catalytic general base is Asp166 in human PFK-M numbering (an analogous Asp591 sits in the C-terminal regulatory domain but is not the catalytic residue) — always check the numbering scheme when cross-referencing the literature.

How to tell a proposal from a fact on screen PROPOSAL markers (amber chip, clickable) + amber dotted phrases = mechanism inferred from crystal geometry, mutagenesis and kinetics, not directly measured: the Asp127 proton-removal path and the γ-P flight & inversion geometry, the residue-chemistry selectivity, and the Mg²⁺ bridging contacts. Everything drawn in motion between crystal states (move-in, crossfade, release direction) is a literature-based reconstruction. Statements without a marker = deposited structure facts (the 4PFK F6P pose, the 3.02 Å / 2.98 Å / 1.89 Å separations, the 1PFK product complex, the bound residue coordinates). No transition state is ever observed.

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: the 4PFK F6P complex pose, 1PFK product pose, Asp127/Arg171 side chains, and the three printed separations (3.02 Å · 2.98 Å · 1.89 Å — each a single value read from the deposited entry; the 3.02 Å and 1.89 Å are declared as geomClaims pfk-asp-o1 / pfk-p1-o1, while the 2.98 Å Arg171 contact carries no declared claim). The inferred layer (PROPOSAL/[schematic]): the proton-hop bookkeeping of Asp127, the in-line S_N2 inversion, the γ-PO₃ flight, the ATP scaffold rivet, and the whole regulatory R↔T dynamic.
Arrow & label conventions Gold ball + arrow = the moving H⁺ (the deprotonated 1-OH proton; schematic sphere, Rule 23). Amber arrow = the γ-PO₃ phosphoryl flight (legend: "flying PO₃ = phosphoryl transfer"). Green Mg–O lines = Mg²⁺ coordination. Å-distance lines = reaction-coordinate markers (3.0 Å label = real measured contact; 3.4 label = assembly seam, flagged in text). Other H atoms are not drawn.
Why is the ATP a "scaffold + γ-PO₃" schematic? A true PFK-1·F6P·MgATP ternary complex has never been crystallized. The deposited adenosine is the product-state ADP (1PFK), which sits in the same N-/P-pocket the ATP uses; we rivet a hand-built terminal γ-PO₃ onto it and call the pair "ATP (γ-P)". The γ-P donor seat ≈ 3.4 Å from O1 is our assembly choice to stage the back-side attack — it is not presented as a crystallographic number and has no geomClaim.
Static protein, reconstructed motion The protein mesh is static — no conformer sampling, thermal motion, or subunit/domain rotation is simulated. What this screen therefore cannot show (see the Biochemist review box): (i) the bacterial closed↔open domain movement (1PFK) and the eukaryotic regulation-domain rotation (8W2G/8W2H) are labels, not deformed meshes; (ii) the homotropic F6P cooperativity and the R↔T effector switch are described, not animated; (iii) the F6P 6-P cradle residue belongs to the partner subunit (Arg162′, chain B), which is not rendered in this single-frame snapshot; (iv) every inter-state transition is "literature-based", not measured. Animations interpolate schematic states only.
Precision vs. accuracy — Å numbers are single crystallographic states The printed separations (3.02 Å Asp127·OD2 ⋯ F6P·O1; 2.98 Å Arg171·NH1 ⋯ F6P·O1; 1.89 Å F1,6BP·P1 ⋯ F6P·O1 footprint) are single values read from the deposited entries; the 3.02 Å and 1.89 Å are declared geomClaims that the Rule 34 audit re-measures from js/data_pfk.js, while the 2.98 Å Arg171 contact is a printed reading that carries no declared claim. They are not measured dynamics and carry crystallographic precision only. The γ-P seat (≈ 3.4 Å) and every rendered carboxylate/arginine contact of the reconstruction are our assembly and carry no printed Å as crystallographic fact.
Physiological regulation — the textbook allosteric checkpoint PFK-1 is the classic allosteric flux gate. Bacterial (this frame): homotropic F6P cooperativity (Hill h ≈ 1.8–2 in the tetramer), allosteric ADP/GDP activation and phosphoenolpyruvate (PEP) inhibition — the "energy charge" sensors of the cell. Eukaryotic: the R↔T tetramer is switched on by AMP and fructose-2,6-bisphosphate, off by ATP and citrate; F-2,6-BP is the dominant physiological activator, and its levels are set by PFKFB3 (EC 2.7.1.105 — a separate bifunctional enzyme, see its page, "not identical with EC 2.7.1.105"). PFK-M is the isozyme most resistant to ATP inhibition, reflecting muscle energy demands.
Kinetics scope — one event, not a timecourse This tool animates a single catalytic event. It does not model timecourses, steady-state kinetics, K0.5 / kcat / V_max · [S] behavior, allosteric state transitions, or cooperative binding dynamics. Use it to learn the chemistry (deprotonation → phosphoryl transfer → product state) and pair it with a kinetics chapter (e.g., Lehninger · Enzymes) for the regulation story.
Sources — bibliography of this page S1 — PDB 4PFK: G. stearothermophilus PFK-1 F6P complex — the substrate pose and the catalytic separations Asp127·OD2 ⋯ F6P·O1 and Arg171·NH1 ⋯ O1 (values on the Mechanism fidelity card; geomClaim pfk-asp-o1) · bacterial regulatory site (homotropic F6P cooperativity, ADP/GDP activation · PEP inhibition) S2 — PDB 1PFK: E. coli PFK-1 reaction-product complex — the ADP scaffold that hosts the drawn ATP and the transferred-phosphate footprint F1,6BP·P1 ⋯ F6P·O1 (value on the Mechanism fidelity card; geomClaim pfk-p1-o1); in-line inverting Sₙ2 + stereochemical assay (Jarvest & Lowe 1981, via Shirakihara & Evans 1988) S3 — Hellinga HW & Evans PR, Nature 327:437, 1987, DOI 10.1038/327437a0, PMID:2953977 — Asp127→Ser directional-loss magnitudes (quoted in the Mechanism fidelity card) pin the general base; associative-vs-dissociative TS debate (blurred in enzymes) S4 — Berger SA & Evans PR, Biochemistry 1992, DOI 10.1021/bi00153a017, PMID:1390710 — Asp127/Asp129 electrostatic pKa lift · Lainé R et al., Eur J Biochem 1992, DOI 10.1111/j.1432-1033.1992.tb17148.x — pH-dependence of catalysis is not the Asp127/Asp129 pair S5 — PDB 8W2G · 8W2H: cryo-EM liver isozyme PFK-L — eukaryotic R↔T regulation-domain rotation, label-referenced only (see "Static protein") S6 — Kinetics context (human PFK-M): Fernandes PM et al., Biochem J 477(22):4425–4441, 2020, DOI 10.1042/bcj20200656 — k_cat, K_0.5(F6P), K_0.5(ATP) and Hill h values quoted in the EvidenceView body; BRENDA EC 2.7.1.11 (P08237) — β-anomer stereospecificity; F-2,6-BP maker = PFKFB3, EC 2.7.1.105, "not identical" S7 — UniProt P08237 (PFK-M) · NCBI Gene 5213 (PFKM); isozymes PFK-M / PFK-L / PFK-P; Tarui disease (PFK-M deficiency) S8 — Repo display conventions: Rule 30 (narrated claims reconstructible from the scene) · Rule 34 geometry (3 geomClaims in tools/manifests/pfk.json) · every Å = a single crystallographic value (Precision vs. accuracy card); the γ-P seat distance (narrated on the ATP scaffold card) is an assembly seam, not a geomClaim · [schematic]/PROPOSAL = inferred from crystal geometry + mutagenesis + kinetics, a PFK TS is never observed

› Backbones, residue side chains and both ligand poses (F6P, and the F1,6BP/ADP/Mg product set) are taken directly from the listed PDB entries (4PFK, 1PFK) superposed into one common frame. F6P is a real crystal ligand — only the ATP is a scaffold + schematic γ-PO₃ (no ternary crystal). Printed Å = single crystallographic values, re-measured by the audit. Where inter-state transitions are stitched, the recomposed coordinates are schematic — see "Precision vs. accuracy" above.