Enolase - 3D Mechanism Simulator

Loading structural data… (2PSN)

References & Databases

Structure Human ENO1 scaffold 2PSN (the entry has no standalone PMID, i.e. "to be published"), 2.20 Å, R = 0.184 / R_free = 0.217, tetragonal P4₂; per chain the deposit holds two Mg²⁺ (MG701 high-affinity \u2192 Asp244·Glu292·Asp317; MG702 loop \u2192 Ser39 + phosphate) and no substrate. Biological unit is a homodimer; the scene shows one chain. The same crystal form is 3B97 (Kang et al., Acta Crystallogr D 64:651–657, 2008, PMID:18560153), which carries SO₄ in place of 2PSN's phosphate.
Metal-site mechanism Mg²⁺ octahedral coordination at the enolase high-affinity metal site: Wedekind JE, Reed GH & Rayment I, Biochemistry 1995, PMID:7703246 (1EBH, yeast enolase — enolase metal/mechanism is highly conserved).
① 2PG docks — chelates the metal site [schematic] 2-phosphoglycerate docks with its carboxylate bidentate-chelating the Mg²⁺ ions (PubChem geometry, schematic), which polarize the substrate and sharpen the C2 proton toward the catalytic lysine. Poses carry explicit H atoms (PubChem); only the C2-H is tracked. (2R) configuration kept per Rhea; stereochemistry unvalidated in poses. Active-site loops close over the pocket on binding (not animated; static mesh).Source: schematic pose — no human ENO1·2-PG co-structure deposited (Why-is-the-substrate card); frame = measured 2PSN backbone + two-Mg metal pocket; ligand = PubChem 3D, (2R) per Rhea, stereochemistry unvalidated
② C2–H abstraction — Mg-stabilized enolate Conserved Lys342 (2PSN author numbering; UniProt P06733 = Lys343) abstracts the C2 proton, leaving the Mg²⁺-stabilized enolate of 2-PG — 2PSN coordinate contacts, measured to the high-affinity Mg (MG701) in this substrate-free (phosphate-only) pocket: Lys342·Nζ to Mg 3.41 Å, Glu209·Oε to Mg 6.92 Å, Lys342–Glu209 6.8 Å (Rule 34 reproduced). These frame the metal site — they are not substrate contacts; the substrate itself is [schematic]. Active fraction is reverse-protonated (Lys-NH2/Glu-COOH, 1P43), so the abstracting base is the neutral amine.Source: measured (2PSN, substrate-free phosphate-only pocket; Rule 34 geomClaims): Lys342·Nζ–Mg(MG701) = 3.41 Å, Glu209·Oε–Mg = 6.92 Å, Lys342–Glu209 = 6.8 Å — not substrate contacts · Lys342-base enolate + reverse-protonated active fraction (Lys-NH₂/Glu-COOH, 1P43) = literature (Metal-site mechanism card)
③ C3–OH leaves — water appears, PEP forms Conserved Glu209 (2PSN = UniProt 210) protonates the departing C3–OH, eliminating water anti to the C2–H — the leaving group is drawn as a water molecule (a real product, never silently dropped) and what remains is PEP. The anti stereochemistry follows the conserved yeast mechanism with the substrate bound: the carboxylate-bridged 2-Mg·substrate geometry and the Lys-base/Glu-proton-donor roles are from the yeast 2PGA-Mg₂ complex 1ONE (Larsen TM et al., Biochemistry 35:4349–4358, 1996, PMID:8605183) — the 1EBH/Wedekind structure is the metal-site reference, not the substrate-step source — transferred to the human scaffold.Source: conserved yeast substrate-step mechanism — Larsen et al. 1996, 2PGA-Mg₂ complex 1ONE: Glu protonates the departing C3–OH, water eliminated anti to C2–H, Lys-base/Glu-proton-donor roles — transferred to the human 2PSN scaffold (no human substrate co-structure)
④ PEP + H₂O released — metals stay home [schematic] PEP exits toward pyruvate kinase; it carries a high phosphate-transfer potential — the high-energy molecule metabolized by PK (a ΔG value “nearly double that of ATP” was not re-verified this review — see Kinetics scope). Water leaves with it; the Mg²⁺ ions stay at the metal site.Source: [schematic] step order — PEP + H₂O leave separately, metals stay resident (Metal-site mechanism card · manifest step 4) · phosphate-transfer potential paid forward to PK; ΔG not re-verified (Kinetics scope card)

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: the human ENO1 backbone of 2PSN with the two Mg²⁺ ions at the metal site, plus the metal–residue contacts quoted in card ②. The inferred layer (all [schematic], cards ①–④): the 2-PG and PEP poses (PubChem geometry re-anchored on the real metal pocket — caveats in card ①), the C2–H abstraction (card ②), and the water-eliminating step to PEP (card ③); together with the reverse-protonated active fraction (Lys-NH2/Glu-COOH, 1P43) and the explicit-H/(2R) bookkeeping disclosed in card ①. Yeast-derived mechanistic elements are transferred onto the human scaffold because the enolase metal/mechanism is highly conserved: the metal octahedral geometry from 1EBH (Wedekind 1995) and the substrate-step stereo/roles (carboxylate-bridged 2-Mg, Lys base, Glu proton donor) from 1ONE (Larsen 1996).
Why is the substrate "[schematic]"? 2PSN is a metal-occupied, substrate-free scaffold — it holds the two Mg²⁺ and a phosphate at the active site; what is missing is only 2-phosphoglycerate (or PEP), and no human ENO1 co-structure bound to either is deposited. Every substrate state is therefore rebuilt from small-molecule geometry (PubChem 3D) re-anchored onto the real metal pocket: carboxylate chelating the two Mg²⁺, the C2–H aimed at Lys342. The backbone, ions and residue side chains are the only measured heavy atoms; explicit hydrogens exist only so the C2 proton can be tracked (bookkeeping in card ①). The tag means exactly this — a real pocket carrying rebuilt, literature-guided ligands.
Physiological regulation & moonlighting context Enolase is not a flux gate — the 2-PG ⇆ PEP step sits near equilibrium and runs backward in gluconeogenesis; downstream pyruvate kinase pulls it forward in glycolysis. Isozymes are tissue-split: ENO1 (α, ubiquitous) · ENO3 (β, muscle) · ENO2 (γ, neuron) as homo/heterodimers. The same polypeptide moonlights: alternate-initiation isoform MBP-1 represses c-myc, surface enolase is a plasminogen receptor, and anti-ENO1 autoantibodies mark several diseases (UniProt P06733; Kang 2008).
Kinetics scope (no measured timecourse on this page) One 2-PG → PEP anti-elimination is drawn, not a steady-state run. Earlier versions quoted compiled reference values — human enolase K_m(2-PG) ≈ 0.3 mM (BRENDA EC 4.2.1.11) and k_cat ≈ 10² s⁻¹ — those numbers are withdrawn pending verification (UniProt P06733 carries no K_m/k_cat for this reaction and the BRENDA entries could not be re-confirmed in the latest review, 2026-09-19). The step runs near equilibrium in the cell; the phosphate potential is paid forward to PK.
Sequence DNA/gene NCBI Gene 2023 · protein UniProt P06733
Sources — bibliography of this page S1 — PDB 2PSN (no standalone PMID, "to be published"; crystal statistics on the deposit record): human ENO1 metal-occupied substrate-free scaffold — MG701 high-affinity (Asp244·Glu292·Asp317), MG702 loop (Ser39 + phosphate), no substrate; measured metal-site contacts (Rule 34 geomClaims, registered in tools/manifests/eno.json): Lys342·Nζ–Mg(MG701), Glu209·Oε–Mg, Lys342–Glu209 — these frame the metal site, they are not substrate contacts S2 — Same crystal form: 3B97 (Kang HJ et al., Acta Crystallogr D 64:651–657, 2008, PMID:18560153), carrying SO₄ in place of 2PSN's phosphate; Kang 2008 also documents the multifunctionality (MBP-1/c-myc repression, plasminogen receptor, anti-ENO1 autoantibodies) S3 — Metal octahedral geometry: Wedekind JE, Reed GH & Rayment I, Biochemistry 1995, PMID:7703246 (1EBH, yeast enolase; metal/mechanism highly conserved) — the metal-site reference, not the substrate-step source S4 — Substrate-step stereo/roles (carboxylate-bridged 2-Mg, Lys base, Glu proton donor, water eliminated anti to C2–H): yeast 2PGA-Mg₂ complex 1ONE, Larsen TM et al., Biochemistry 35:4349–4358, 1996, PMID:8605183 — transferred to the human 2PSN scaffold (no human ENO1·ligand co-structure) · reverse-protonated active fraction (Lys-NH₂/Glu-COOH) per 1P43 · mechanism E1cb stepwise rendered, concerted E2 recorded as the opposing view (M-CSA enolase) S5 — Kinetics: the compiled values quoted in the EvidenceView body (human enolase K_m(2-PG), BRENDA EC 4.2.1.11; k_cat) are withdrawn pending verification (UniProt P06733 carries no K_m/k_cat for this reaction; review 2026-09-19) S6 — Sequence: NCBI Gene 2023 · UniProt P06733 (2PSN author numbering = UniProt − 1: Lys342/Glu209 on screen = Lys343/Glu210) S7 — Repo display conventions: Rule 30 (S_N2/elimination cartoons, TS unobserved) · Rule 34 geometry (three geomClaims in tools/manifests/eno.json, every Å a single crystallographic value) · [schematic] = 2-PG / PEP substrate poses (PubChem 3D re-anchored on the real metal pocket — no human ENO1 co-structure bound to either is deposited) · explicit-H / (2R) bookkeeping · Mg²⁺/PO₄ from 2PSN are the only deposited ligands

› Backbones and ligand poses are taken directly from the listed PDB entries, superposed into one common frame. Where no crystalline state exists for a step (e.g., Michaelis geometry), the pose is drawn schematically using PubChem 3D geometries and is labeled as such in the narration.

N-domain(β/α) barrel domainONPMg2+