Fumarase (Fumarate Hydratase) - 3D Mechanism Simulator

Loading structural data… (5UPP)

References & Databases

START HERE — how to read fumarate + H₂O → L-malate A single hydration cartoon on the human fumarase pocket. Real crystal: 5UPP (1.8 Å) with the catalytic RTHTQ loop (Arg233·Thr234·His235 ★·Thr236·Gln237). Biological assembly is a homotetramer; M-CSA 569: the catalytic site spans three of four subunits — not drawn. 5UPP is not ligand-free: it holds HEPES (EPE) at Domain 3 / Lys467 (Ajalla Aleixo 2019) — a crystallization buffer, not a substrate. No fumarate, malate, or catalytic-water coordinates are in this data path — every substrate/water/product pose is [schematic]. Steps ③ and ⑤ are teaching overlays: His235 ★ is shown taking a water proton (gold H⁺); the resulting OH⁻ attacks C2 (blue arrow = nucleophile, not hydride). The catalog acid on the reverse (dehydration) is Ser365 (M-CSA Ser318); Lys371 (M-CSA Lys324) is named as the carbanion electrostatic stabilizer. Glu378 is named, not drawn. Analog co-crystal 1FUQ is named, not drawn. No Å is claimed for substrate contacts.
Crystal structure PDB 5UPP — human fumarate hydratase, 1.8 Å, Ajalla Aleixo MA et al., FEBS J 2019, PMID:30761759 · DOI:10.1111/febs.14782. Biological assembly is a homotetramer; this slice is not that tetramer. The RTHTQ loop (Arg233-Thr234-His235-Thr236-Gln237) is the catalytic loop. The deposited ligand is HEPES (EPE) at Domain 3 / Lys467 — a crystallization buffer, not a substrate, and not asserted here as an allosteric effector. No fumarate, malate, or catalytic-water coordinates are in this data path, so those poses are schematic. Analog co-crystal: 1FUQ (E. coli fumarase C + 3-trimethylsilylsuccinate, Weaver 1996) — named, not drawn.
Mechanism — anti-addition; His base, Ser acid (dehydration catalog) Bekker M et al., FEBS J 2018, PMID:29573314 — stereospecific trans (anti) hydration of fumarate to L-malate; a reversible lyase. M-CSA 569 (E. coli class II, P05042, PDB 1FUQ) writes the dehydration / E1cB direction: His188 activates water to abstract C3-H; Ser318 is the acid that protonates the departing C2-OH; Lys324 is the carbanion electrostatic stabilizer; Glu331 raises His pKa. Human map: His188→His235 ★, Ser318→Ser365, Lys324→Lys371, Glu331→Glu378 (UniProt P07954 numbering). This page draws a hydration cartoon: steps ③ and ⑤ are teaching overlays, not the catalog reverse. Aci-carboxylate is elimination-path language, not a stable hydration intermediate. A concerted one-throw addition is the recorded opposing view to E1cB. Source: M-CSA 569 · Bekker 2018 (PMID:29573314) · Ajalla Aleixo 2019 (PMID:30761759)
0 — Setup: the RTHTQ pocket The catalytic RTHTQ loop presents His235 ★ as the water-activating residue (M-CSA His188). Biological assembly is a homotetramer; the catalytic site spans three of four subunits (M-CSA 569) — not drawn. 5UPP is not ligand-free (HEPES at Lys467) but this data path has no substrate or catalytic water, so the pocket is shown empty for the catalytic story. Opaque, run:null (no autoplay — inspect the pocket first). Ser365, Glu378 and Lys371 are named, not drawn.Source: PDB 5UPP (1.8 Å), Ajalla Aleixo 2019 · M-CSA 569
① — Fumarate binds (C2=C3 into the pocket) [schematic] Fumarate docks with its trans C2=C3 bond across the pocket, olefin opposite His235 ★. Carboxylate contacts from the class-II literature (E. coli 1FUQ) are named, not drawn on this 5UPP slice. No fumarate ligand is in 5UPP — this seat is schematic, and no substrate Å is claimed. Analog co-crystal 1FUQ (3-trimethylsilylsuccinate) is named, not drawn.Source: schematic pose (no fumarate in 5UPP; analog 1FUQ named)
② — Catalytic water binds the attacking face [schematic] A catalytic water slides in above the C2=C3 face. This hydration cartoon aims H2 at His235 ★ NE2; O–H1 is the nucleophile in the next overlay. No catalytic-water coordinates are in this 5UPP data path. The catalog reverse of E1cB would have Ser365 as the base on the adding water (not drawn). (hypothetical, lit.-based)Source: water-activating His, M-CSA 569
③ — His235 ★ abstracts the water H⁺ (water-activating base) (hypothetical, lit.-based) A gold H⁺ sphere rides a gold arrow from the water H2 to His235 ★ NE2 and parks there — it is not absorbed. Removing H2 leaves O–H1 = OH⁻, the nucleophile. This nucleophile-generation assignment is a teaching overlay — the catalog reverse of E1cB would have Ser365 as the base on the adding water (not drawn). His235 is the water-activating residue (M-CSA His188), not the catalog acid. Glu378 is named as the His pKa partner, not drawn. The resident fumarate is dimmed (ghost) so the proton path reads.Source: M-CSA 569 · Bekker 2018
④ — OH⁻ attacks C2 → L-malate body (hypothetical, lit.-based) The OH⁻ (blue arrow = nucleophile lone pair, not a hydride) attacks C2 from its face — an anti (trans) addition. A brief C3-carbanion overlay is a teaching label. Aci-carboxylate is elimination-path language (malate → fumarate, M-CSA 569) and is not drawn as a stable hydration intermediate. The fumarate skeleton cross-fades in place into the L-malate scaffold. Slow-motion on purpose.Source: anti-addition, Bekker 2018 (PMID:29573314) · aci-carboxylate warning, M-CSA 569
⑤ — H⁺ to C3 (teaching overlay) → L-malate complete (hypothetical, lit.-based) A teaching overlay returns the parked gold H⁺ from NE2 to C3 (second gold arrow) so the screen closes L-malate (2S). This is not the M-CSA 569 acid: on the dehydration path the catalog acid is Ser365 (M-CSA Ser318), which protonates the departing C2-OH — Ser365 is not drawn. Net on this page: water added OH to C2, H to C3 from opposite faces.Source: teaching overlay vs M-CSA 569 Ser acid
⑥ — L-malate released — on to MDH [schematic] L-malate leaves toward malate dehydrogenase (TCA step 8). The enzyme returns to the empty catalytic pocket. The reaction is fully reversible — fumarase also dehydrates malate; M-CSA 569 is written in that direction.Source: reversible lyase, EC 4.2.1.2 · next TCA step MDH

How to read this page — a first-pass primer First pass (ignore the noise): watch the order — Setup (RTHTQ pocket, His235 ★) → ① fumarate docks C2=C3 across the pocket → ② a water docks, H2 toward His235 → ③ gold H⁺ goes water H2 → His235 NE2 (water becomes OH⁻) → ④ blue OH⁻ (nucleophile, not hydride) attacks C2, fumarate becomes malate → ⑤ teaching overlay: parked H⁺ to C3 → ⑥ L-malate leaves. Second pass (the numbers): no Å are printed at all — 5UPP has no fumarate/malate/catalytic-water coordinates in this data path; every substrate/water/product pose is disclosed [schematic]. 5UPP does hold HEPES at Lys467. Third pass: open “Mechanism fidelity” and “Disagreements” to separate the measured crystal frame from the literature-based reconstruction, and M-CSA 569 (dehydration, Ser acid) from this hydration overlay.
Chemistry primer — hydration, Markovnikov-free, anti addition Fumarate is the trans C4 dicarboxylate. Adding water across the C=C gives malate; the enzyme adds OH to C2 and H to C3 from opposite faces, so the product is the (2S) enantiomer. On this page His235 is shown as the water-activating residue in a hydration cartoon (steps ③ and ⑤ are overlays). The catalog acid on the reverse (dehydration) is Ser365 (M-CSA Ser318), not drawn. Aci-carboxylate names the elimination-path carbanion (malate → fumarate), not a stable hydration intermediate. The gold/blue balls are trajectory cartoons, not curly-arrow electron pushing. Blue here is OH⁻ (nucleophile), not hydride. Net: fumarate + H₂O ⇄ L-malate.
Words used on screen — OH⁻ · anti addition · teaching overlay OH⁻ — the hydroxide nucleophile left after His235 removes one water proton; it attacks C2. The shared legend’s blue arrow means hydride/electron on other pages; on this page blue = OH⁻ nucleophile. Anti (trans) addition — the two added groups (OH and H) end up on opposite faces of the former C=C. Consumed water — the water is a reactant: its O becomes the C2-OH and one of its H becomes the C3-H. Aci-carboxylate is not used as a hydration-intermediate name on screen — it is elimination-path language (see Disagreements).

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: the 5UPP frame (1.8 Å) with the RTHTQ loop (His235 ★ drawn). The deposited ligand is HEPES (EPE) at Domain 3 / Lys467 — not a substrate, not drawn. Biological assembly is a homotetramer; this slice is not that tetramer. The inferred layer (all [schematic]): the fumarate, water and L-malate poses (no catalytic-water or substrate coordinates in this data path; analog 1FUQ named), and steps ③ and ⑤ as teaching overlays (M-CSA 569 writes dehydration / E1cB with Ser as the acid). No Å label is drawn for any substrate contact. Ser365, Glu378 and Lys371 are named, not drawn. The star marks His235 ★; Arg233/Thr234/Thr236/Gln237 are context only.
Disagreements on record (a) Concerted vs stepwise / E1cB. The screen splits deprotonation, attack and proton delivery into separate events as a hydration cartoon. M-CSA 569 is written as dehydration / E1cB; a concerted one-throw addition is the recorded opposing view and is not drawn. (b) Identity of the acid. M-CSA 569 (dehydration, E. coli) assigns His188 as the water-activating residue and Ser318 as the acid that protonates the departing C2-OH. This page’s steps ③ and ⑤ are teaching overlays, not that catalog reverse. Human map: His188→His235, Ser318→Ser365, Lys324→Lys371, Glu331→Glu378. (c) Aci-carboxylate. That name belongs to the elimination path (malate → fumarate); it is not drawn here as a stable hydration intermediate. (d) Substrate numbering. C2/C3 are teaching labels on a schematic pose (fumarate has no deposited atom names in 5UPP). (e) Direction. Fumarase is fully reversible; the page draws hydration; M-CSA 569 is written as dehydration. (f) HEPES at Lys467. Occupancy of a crystallization buffer is a crystal fact; an allosteric-effector claim is not asserted here.
Regulation — Lys467 / HEPES · FH tumor suppressor Ajalla Aleixo 2019 found HEPES (EPE) bound at Domain 3 through a set of contacts that includes Lys467. That paper reports a crystallization-buffer occupancy and discusses whether the site can affect kinetics; this page does not assert HEPES as an allosteric effector (named, not drawn). Fumarase is also a tumor-suppressor (FH). Loss-of-function FH mutations cause fumarase deficiency and hereditary leiomyomatosis/renal-cell cancer, with fumarate accumulation acting as an oncometabolite that inhibits prolyl hydroxylases. The mitochondrial and cytosolic isoforms share this active-site chemistry. Named in text, not drawn as mutant structures.Source: Ajalla Aleixo 2019 (PMID:30761759) · FH genetics (OMIM 136850), named not simulated
Kinetics scope (no measured timecourse on this page) One hydration event is drawn, not a steady-state run. Compiled BRENDA values for EC 4.2.1.2 are not re-asserted here — no measured k_cat/K_m on this page [schematic]; the page is qualitative-with-grounds (order-of-magnitude only). Pair with a kinetics chapter.Source: BRENDA EC 4.2.1.2 — no k_cat/K_m re-asserted
Sequence Human FH (mitochondrial/cytosolic dual-targeted) UniProt P07954 · NCBI Gene 2271 · fumarase deficiency / tumor-suppressor relevance at OMIM 136850.
Sources — bibliography of this page S1 — PDB 5UPP: human fumarate hydratase, 1.8 Å; Ajalla Aleixo MA et al., FEBS J 2019, PMID:30761759 · DOI:10.1111/febs.14782 — RTHTQ loop; HEPES (EPE) at Domain 3 / Lys467 (not ligand-free; not a substrate co-crystal) S2 — Mechanism: M-CSA 569 (E. coli class II, P05042, PDB 1FUQ) — dehydration / E1cB: His188 activates water; Ser318 is the acid on the departing C2-OH; Lys324 is the carbanion electrostatic stabilizer; Glu331 raises His pKa. Human map His188→His235, Ser318→Ser365, Lys324→Lys371, Glu331→Glu378. Analog co-crystal 1FUQ (Weaver 1996, PMID:8909293) — named, not drawn S3 — Anti-addition stereochemistry: Bekker M et al., FEBS J 2018, PMID:29573314 — stereospecific trans (anti) hydration of fumarate to L-malate; reversible lyase; individual steps not directly observable — “(hypothetical, lit.-based)” S4 — Sequence: UniProt P07954 (human FH) · NCBI Gene 2271 · fumarase deficiency / tumor-suppressor — OMIM 136850 S5 — Kinetics: BRENDA EC 4.2.1.2 — no k_cat/K_m re-asserted on this page

› Backbones and catalytic loops are taken directly from the listed PDB entries, superposed into one common frame. 5UPP holds HEPES (EPE) at Lys467, not a substrate; fumarate/water/malate poses are drawn schematically and labeled as such in the narration.

Catalytic domainC-terminal domainONOH⁻ (blue arrow)