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Structure — the real phospho-primed frame · 4GPZ
Human PGAM1 (UniProt P18669, residues 2–243 on screen, PDB-author = UniProt numbering) with the catalytic His11 covalently phosphorylated on its ring Nε2 — the crystallographic NEP ligand (NE2 ⋯ P = 1.68 Å; the other ring N, ND1 ⋯ P = 3.72 Å; UniProt ACT_SITE 11 calls the state Tele-phosphohistidine while the PDB MODRES spells it “N1-phosphonohistidine” — two naming conventions, treat the coordinate bond, not the label, as the evidence), res. 1.65 Å (monoclinic P 212121; phospho-His11 occupancy 0.70): RCSB 4GPZ (Hitosugi T et al., Nat Commun 4:1790, 2013; PMID:23653202 — the structure paper; the same paper deposits 4GPI, the unphosphorylated apo form at 2.08 Å. The companion tumor-metabolism paper is the Cancer Cell 22:585, 2012 / PMID:23153533, cited in card ④). This is the state the enzyme starts from every turnover — not the dephospho-apo form. The 2F90 structure (human BPGM — the RBC 2,3-BPG-synthesizing sister enzyme, PDB 2F90 holds 3-PG + AlF₄⁻, Wang et al., J Biol Chem 281:39642–39648, 2006, PMID:17052986) is not this enzyme's frame: every protein Å here comes from 4GPZ alone. Shown as a monomer (chain A); the biological unit is a homodimer (UniProt P18669).
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① Phospho-His11 · the measured crystal contacts
His11 Nε ⋯ P = 1.68 Å (NEP, covalent N–P bond, measured from the deposited coordinates — the page’s first geomClaim) · the phosphate is held by the His11 cradle: Arg62 NE ⋯ O2P = 2.92 Å, Arg62 NH2 ⋯ O2P = 3.24 Å, Glu89 OE2 ⋯ O2P = 2.60 Å, Arg10 NH2 ⋯ O1P = 2.97 Å, His186 ND1 ⋯ O2P = 2.85 Å and Gly187 N ⋯ O3P = 2.63 Å (all measured, re-checked under Rule 34 geomClaims). The cradle is the anionic-TS cushion of both displacements. Per M-CSA 504 (yeast Glu86), the Glu89 equivalent is additionally a general acid/base proton shuttle for the transfers — drawn here as cradle only; transient proton moves are not shown (net change: none).Source: measured from the PDB 4GPZ deposited coordinates (six geomClaims registered in
tools/manifests/pgam.json, Rule 34): His11 Nε ⋯ P = 1.68 Å · Arg62 NE/NH2 ⋯ O2P = 2.92/3.24 Å · Glu89 OE2 ⋯ O2P = 2.60 Å · Arg10 NH2 ⋯ O1P = 2.97 Å · His186 ND1 ⋯ O2P = 2.85 Å · Gly187 N ⋯ O3P = 2.63 Å; structure paper Hitosugi et al. 2013 (Nat Commun 4:1790); Glu89 proton-shuttle role: M-CSA 504 (yeast Glu86), drawn as cradle only
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② 3-PG / 2-PG / 2,3-BPG poses — all [schematic]
4GPZ carries no substrate — to date no human PGAM1 structure with 3-PG bound is deposited (4GPI, same paper, is the apo form; the yeast PGAM1·3-PG ternary 1QHF, 1.7 Å, grounds the M-CSA record used on this page). The bound-3-PG conformation on ② reuses the citrate seat of the citrate-inhibited human enzyme 1YFK (Wang Y et al., Biochem Biophys Res Commun 331:1207, 2005; PMID:15883004) transfered onto the 4GPZ frame; the C2-OH “reach” distance O2H ⋯ P ≈ 6.1 Å is a pose target, not a claimed geometry — it closes across the displacement. The 2,3-BPG clamp on ③/④ (both phosphates together) is, likewise, a disclosed schematic of crowding (near contacts ≈1.9–2.4 Å, never interpenetrating). Poses keep the (2R) glycerate configuration but stereochemistry is unvalidated in schematic coordinates; phosphate charges (dianionic at pH 7) are likewise not drawn.Source: registered schematic poses, not measured — 4GPZ carries no substrate and no human PGAM1·3-PG co-crystal is deposited (4GPI = apo; yeast PGAM1·3-PG ternary 1QHF, 1.7 Å, grounds the M-CSA record); the bound-3-PG skeleton reuses the citrate seat of 1YFK (Wang et al. 2005) transferred onto the 4GPZ frame; O2H ⋯ P ≈ 6.1 Å = pose target, not a claimed geometry (step-1 label in
tools/manifests/pgam.json); the 2,3-BPG clamp on the transfers ≈ 1.9–2.4 Å = disclosed near-contact schematic, never interpenetrating
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③ Ping-pong — double in-line phosphoryl transfer
The classic ping-pong mechanism (family record — a specific modern citation was not re-verified this review; the classic steady-state kinetics are the 1960s–70s papers): (1) His11–P is attacked by the substrate C2-OH (O(C2) ⋯ P ⋯ Nε in-line) → 2,3-BPG; (2) the intermediate’s C3-P re-attacks His11 (same in-line geometry) → 2-PG + re-primed E~P. In cells the intermediate may dissociate between half-reactions and rebind (shown bound for clarity). The CO₃⁻/phosphate separation paradigm positions the two glyceryl phosphates; both displacements use inversion at P (phosphoryl transfer, not “phosphohistidine relay” crudely — the proton inventory is unchanged; the dibasic phosphate group itself is the covalently-migrating unit, drawn by the flying PO₃ groups in steps ② and ③; contrast the cofactor-independent iPGM class (EC 5.4.2.12, Mn2+-dependent phospho-Ser intermediate) — separate genes, not PGAM1 isoforms).Source: ping-pong double in-line displacement = standard family mechanism (classic steady-state kinetics 1960s–70s; Winograd et al., J Mol Biol 268:248, 1997, PMID 9149146 — registry REACTION_EQUATIONS.md ## pgam, not re-verified this review; M-CSA record) — both C2- and C3-P displacements use inversion at P, [schematic], no TS observed; the cofactor-independent iPGM class (EC 5.4.2.12, Mn²⁺-dependent phospho-Ser intermediate) is a separate gene family, not a PGAM1 isoform
Legend & Fidelity
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Mechanism fidelity — measured vs. inferred
The measured layer: the 4GPZ chain with the NEP phospho-His11 and its cradle of contacts — card ① is entirely measured (the Å figures shown on screen are deposited-coordinate values re-checked against the PDB; full list in the drawerNote). The inferred layer (all [schematic]): the three substrate/intermediate poses — card ②, because no human PGAM1 3-PG co-crystal is deposited and the C2-OH reach (≈6.1 Å) is targeted, not claimed — and the ping-pong double in-line inversion geometry of the two handoffs (card ③, family record). Two things are framed rather than drawn: the Glu89 equivalent M-CSA 504 proton shuttle (transient proton moves are net-zero and thus undisclosable by motion) and the dianionic phosphate charge state at pH 7. Both are genuine chemistry the animation cannot exhibit, so they stay as disclosed context. The protein frame itself is a single deposited species with no superposition seam (drawerNote).
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④ Physiological regulation & moonlighting context
PGAM1 is not a flux gate — the 3-PG ⇄ 2-PG equilibrium sits near unity under cellular conditions and the downstream enolase keeps pulling 2-PG → PEP, which is what actually drives glycolysis forward here (a specific ΔG°′ figure was not re-verified this review — see Kinetics scope). The same enzyme moonlights: knockdown slows growth of many tumor lines and is derepressed by loss of TP53 (PGAM1 is a negative transcriptional target of TP53) (Hitosugi T et al., Cancer Cell 22:585, 2012; PMID:23153533). Its catalytic priming 2,3-BPG is catalytic (trace amounts that stoichiometrically dephosphorylate once per site, then recirculate in the reverse direction) — summarized as “2,3-BPG priming” in the build frame. Acute regulation (cards only, not animated): Y26 phosphorylation releases autoinhibitory E19 and stabilizes the active H11-phosphorylated conformation (Nat Commun 2013, PMID:23653202); SIRT1 deacetylation (K253/K254) tunes activity to glucose availability (JBC 2011).Source: moonlighting + TP53 derepression: Hitosugi T et al., Cancer Cell 22:585, 2012, PMID 23153533 (plain text — no link for it on this page); Y26 phosphorylation releases the autoinhibitory E19 and stabilizes the active H11-phosphorylated conformation: Nat Commun 4:1790, 2013; near-unity 3-PG ⇄ 2-PG + downstream enolase pull + catalytic 2,3-BPG priming: this regulation card + "Kinetics scope" card — no specific ΔG°′ was re-verified this review
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Kinetics scope (no measured timecourse on this page)
One turnover is two phosphate handoffs on His11. Earlier versions quoted compiled reference values — PGAM1 kcat ≈ 200–500 s⁻¹ (BRENDA EC 5.4.2.11; assay-dependent), Km(3-PG) ≈ 0.1–0.5 mM, 2,3-BPG Km ≈ 0.3 µM (cofactor mode), 2-PG Km similar magnitude, and ΔG°′ ≈ +4.4 kJ·mol⁻¹ with “near-unity” equilibrium — those numbers are withdrawn pending verification (UniProt P18669 carries no Km/kcat for this reaction, and the BRENDA entries could not be re-confirmed in the latest review, 2026-09-19; the near-unity/prime-the-enzyme narration is kept on textbook grounds). Activation requires a priming 2,3-BPG molecule (or an equivalent phosphate donor); the dephospho-enzyme is essentially dead.
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Residue numbering & databases
Human PGAM1, PDB-author 2–243 = UniProt P18669. Catalytic/marking: His11 ★ (phospho-acceptor), Arg62 ★ & Glu89 ★ (phosphate cradle). The same His-phosphotransfer + Arg/Glu cradle chemistry is shared by the whole 2,3-BPG-dependent family — PGAM1 (a.k.a. PGAM-B), PGAM2 (muscle isozyme) and BPGM (RBC, synthesizes 2,3-BPG); UniProt gives no distinguishing catalytic motif, so the page does not claim one. Arg10 · His186 · Gly187 complete the cradle. DNA/gene NCBI Gene 5223 · protein UniProt P18669 · EC 5.4.2.11 (BRENDA registry). Numbering is PDB-author = UniProt canonical (initiator Met = 1, hence His11); mature-chain numbering (Met cleaved) calls the same residue His10 (e.g., ExplorEnz).
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Sources — bibliography of this page
S1 — PDB 4GPZ: human PGAM1 phospho-His11/NEP structure (single species, no superposition seam); Hitosugi T et al., Nat Commun 4:1790, 2013, PMID:23653202 — six geomClaims registered in
tools/manifests/pgam.json (His11 Nε⋯P, Arg62 NE/NH2⋯O2P, Glu89 OE2⋯O2P, Arg10 NH2⋯O1P, His186 ND1⋯O2P, Gly187 N⋯O3P — Å values shown on the ① card and the drawerNote)
S2 — Substrate poses are [schematic]: 4GPZ carries no substrate and no human PGAM1·3-PG co-crystal is deposited (4GPI = apo, same paper); the bound-3-PG skeleton reuses the citrate seat of 1YFK (Wang Y et al., Biochem Biophys Res Commun 331:1207, 2005, PMID:15883004) transferred onto the 4GPZ frame; the yeast PGAM1·3-PG ternary 1QHF (1.7 Å) grounds the M-CSA record 504 (yeast Glu86 = human Glu89 proton-shuttle context); O2H ⋯ P reach = pose target, not a claimed geometry (step-1 label in pgam.json)
S3 — Mechanism: classic ping-pong double in-line phosphoryl transfer — steady-state kinetics of the 1960s–70s; Winograd E et al., J Mol Biol 268:248, 1997, PMID:9149146 (registry REACTION_EQUATIONS.md ## pgam, not re-verified this review; M-CSA record) — both C2- and C3-P displacements invert at P; the cofactor-independent iPGM class (EC 5.4.2.12, Mn²⁺-dependent phospho-Ser intermediate) is a separate gene family
S4 — Regulation & moonlighting: TP53-negative-target derepression — Hitosugi T et al., Cancer Cell 22:585, 2012, PMID:23153533 (plain text on this page) · Y26 phosphorylation releases autoinhibitory E19 and stabilizes the active H11-phosphorylated form (Nat Commun 4:1790, 2013 = PMID:23653202) · SIRT1 deacetylation of K253/K254 (JBC 2011) · catalytic 2,3-BPG priming
S5 — Kinetics: the compiled reference values quoted in the EvidenceView body (PGAM1 kcat, Km(3-PG), 2,3-BPG Km, ΔG°′) are withdrawn pending verification — UniProt P18669 carries no Km/kcat for this reaction, BRENDA entries not re-confirmed (review 2026-09-19)
S6 — Sequence & databases: NCBI Gene 5223 · UniProt P18669 · EC 5.4.2.11 (BRENDA); PDB-author 2–243 = UniProt P18669 (initiator Met = 1, hence His11; mature-chain numbering calls the same residue His10, e.g. ExplorEnz); family-wide His-phosphotransfer + Arg/Glu cradle shared by PGAM1/PGAM2/BPGM, no isoform-specific motif claimed
S7 — Repo display conventions: Rule 30 (flying PO₃ handoff cartoons, no TS observed) · Rule 34 geometry (six geomClaims in tools/manifests/pgam.json, every Å a single measured value from the deposited 4GPZ coordinates) · [schematic] = 3-PG / 2,3-BPG / 2-PG poses, O2H ⋯ P reach target, 2,3-BPG clamp crowding disclosed (values on the drawerNote; never interpenetrating) · single species (4GPZ), no superposition seam
› The protein frame and the phospho-His11/NEP ligand are 4GPZ deposited coordinates (single species, no superposition seam). Substrate/intermediate/product poses (3-PG, 2,3-BPG, 2-PG, the two phosphate handoffs) are [schematic] reconstructions, each labeled and disclosed. On-screen Å values: 1.68 / 2.92 / 2.60 / 2.97 Å are measured crystal contacts (Rule 34 geomClaims); O2H ⋯ P ≈ 6.1 Å is a schematic reach; the ≈1.9–2.4 Å 2,3-BPG clamp crowding is disclosed, never interpenetrating.