Mitochondrial 3-ketoacyl-CoA thiolase - 3D Mechanism Simulator

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References & Databases

START HERE — how to read acetoacetyl-CoA + CoA → 2 acetyl-CoA A thiolase cartoon on the human ACAA2 frame that actually exists: 4C2J (2.00 Å X-ray) chain A with crystal free CoA. Occupancy is free CoA, not 3-ketoacyl-CoA — never read CoA as the 3-keto substrate. Cys92 on this crystal is CSO (S-hydroxycysteine), not acetyl-Cys. Teaching Cys92 omits the extra OD. Cys92 ★ is the nucleophilic cysteine (UniProt ACT_SITE). His352 is named, not starred. Cys382 is the proton partner. Covalent E~acetyl is compiled and disclosed — not a pure A-page. C4 teaching yields two acetyl-CoA. Blue is nucleophile, not hydride. ACAA1 is another gene. Measured Å are deposited 4C2J free-CoA pairs only.
Crystal / X-ray structures PDB 4C2J — human mitochondrial 3-ketoacyl-CoA thiolase + CoA, 2.00 Å X-ray (Kiema 2014). Coordinates are public. Occupancy is free CoA. Cys92 is CSO. Human ACAA2 UniProt P42765.
Mechanism — Cys92 nucleophile; Cys382 proton partner UniProt P42765 EC 2.3.1.16: a 3-oxoacyl-CoA is cleaved by CoA to an acyl-CoA (n−2) and acetyl-CoA. On this mesh the nucleophilic cysteine is Cys92. His352 is named, not starred. Covalent acetyl-Cys92 is compiled, not this occupancy. ACAA1 is another gene. Source: UniProt P42765 · Kiema 2014
0 — Setup: empty thiolase pocket 4C2J occupancy is free CoA, not 3-ketoacyl-CoA. Cys92 is CSO, not acetyl-Cys. The pocket is shown empty so binding can start. Opaque, run:null (no autoplay). Cys92 ★ is the nucleophile. His0 is an expression tag. Gap 210–213. ACAA1 is another gene.Source: PDB 4C2J (2.00 Å X-ray)
① — Acetoacetyl-CoA binds [schematic] Acetoacetyl-CoA docks on the 4C2J CoA pose — not a deposited 3-keto. CoA on 4C2J is free CoA, not the 3-keto substrate. Overlay Å are not geomClaims. Teaching chain is C4.Source: 4C2J CoA pose + 1F0Y CAA thiol-end overlay
② — Cys92 attacks C3 → acetyl-enzyme + acetyl-CoA (hypothetical, lit.-based) Blue nucleophile rides from Cys92 SG onto schematic acetoacetyl C3. Acetoacetyl-CoA cross-fades to schematic acetyl-CoA (first product) and a compiled acetyl-Cys92 stub. Covalent E~acetyl is disclosed — not a pure A-page. This crystal has CSO, not acetyl-Cys. Slow-motion on purpose. Blue = nucleophile, not hydride.Source: compiled thiolase ping-pong · UniProt P42765 Cys92
③ — First acetyl-CoA leaves The original CoA, now acetyl-CoA (C1–C2 of acetoacetyl), departs. Compiled acetyl-Cys92 stays. C4 teaching makes both products acetyl-CoA; they are not the same molecule. Cys92 is CSO on 4C2J, not acetyl-Cys.Source: thiolase ping-pong bookkeeping
④ — CoA binds Crystal CoA docks — deposited 4C2J occupancy, the second substrate, not a 3-keto. Cys92 SG ⋯ CoA S1P = 3.98 Å is the deposited free-CoA contact, not an acetyl-enzyme geometry.Source: PDB 4C2J CoA
⑤ — CoA-S attacks acetyl-Cys → acetyl-CoA (hypothetical, lit.-based) Blue nucleophile rides from CoA S1P onto compiled acetyl-Cys C. CoA + acetyl stub cross-fade to schematic acetyl-CoA (second product). Cys382 is the compiled proton partner; His352 is named, not starred. Cys92 SG ⋯ His352 NE2 = 3.24 Å is deposited 4C2J. Slow-motion on purpose. Blue = nucleophile, not hydride.Source: compiled thiolase · 4C2J Cys92 / His352
⑥ — Second acetyl-CoA leaves — spiral complete The second acetyl-CoA departs. C4 teaching yields two acetyl-CoA from one spiral turn. This page does not condense (EC 2.3.1.9) and does not run ACAA1. Cys92 is CSO on 4C2J, not acetyl-Cys. Overlay Å are not geomClaims.Source: β-oxidation bookkeeping · EC 2.3.1.16

How to read this page — a first-pass primer First pass: Setup (empty pocket, Cys92 ★, CSO not acetyl) → ① schematic acetoacetyl docks on CoA pose → ② blue Cys92-S → C3, fade to acetyl-CoA + acetyl-Cys → ③ first acetyl-CoA leaves → ④ crystal CoA docks → ⑤ CoA-S → acetyl-Cys, fade to second acetyl-CoA → ⑥ leave; spiral complete. Second pass: 3.98 / 3.24 / 5.79 Å are crystal facts on 4C2J free-CoA complex; 3-keto and acetyl-Cys are [schematic]; CoA is free CoA, not substrate. Third pass: open “Disagreements” for CSO vs Cys, free CoA vs 3-keto, and ACAA1 vs this gene.
Chemistry primer — ping-pong thiolase ACAA2 Cys92 attacks the 3-keto carbon, releasing the original CoA as acyl-CoA(n−2) and leaving an acetyl-enzyme. Incoming CoA then thiolyses that acetyl-Cys to acetyl-CoA. C4 teaching makes both products acetyl-CoA. Blue is nucleophile, not hydride. This is not ACAA1 (peroxisomal) and not condensation EC 2.3.1.9.
Words used on screen — free CoA · CSO · Cys92 ★ · compiled acetyl-Cys Free CoA — 4C2J occupancy, the second substrate / product analog, not 3-ketoacyl-CoA. CSO — Cys92 is S-hydroxycysteine on this crystal; teaching omits OD. Cys92 ★ — nucleophile on this PDB = UniProt. Compiled acetyl-Cys — covalent intermediate, not this occupancy.

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: 4C2J (2.00 Å X-ray) human ACAA2 + free CoA; Cys92 SG ⋯ CoA S1P = 3.98 Å; Cys92 SG ⋯ His352 NE2 = 3.24 Å; Cys92 SG ⋯ Cys382 SG = 5.79 Å. The inferred layer ([schematic]): acetoacetyl-CoA on the CoA pose, transferring nucleophile spheres, acetyl-Cys stub, two acetyl-CoA products. Binding order is compiled staging, not a kinetic fit. The protein mesh is static. Free-CoA Å are not 3-keto geomClaims. Cys92 is CSO, not acetyl-Cys.
Disagreements on record (a) Occupancy vs substrate. 4C2J holds free CoA, not 3-ketoacyl-CoA. (b) CSO vs Cys. Cys92 is S-hydroxycysteine on this crystal — not acetyl-Cys and not unmodified Cys. Teaching omits OD. (c) Covalent acetyl-Cys92 is compiled identity, not this occupancy. (d) His352 is named, not starred. (e) Concerted vs split nucleophile/thiolysis — this page splits ② then ⑤ for 1-event/1-step teaching. (f) ACAA1 is peroxisomal, another gene. (g) Condensation EC 2.3.1.9 and hydrolase EC 3.1.2.- are not this page. (h) His0 expression tag and gap 210–213 are not catalytic. (i) C4 teaching → two acetyl-CoA; longer chains would leave acyl-CoA(n−2) ≠ acetyl-CoA.
Regulation — isoforms · tetramer This spiral is monofunctional ACAA2 / T1 only. Peroxisomal thiolase (ACAA1) is another gene — not this mesh. Tetramer assembly is not this cartoon. Matrix localization is assumed, not a catalytic event on this page.Source: UniProt P42765 · PDB 4C2J · Kiema 2014
Kinetics scope (no measured timecourse on this page) One acetoacetyl-CoA + CoA → 2 acetyl-CoA event is drawn, not a steady-state run. Compiled BRENDA values for EC 2.3.1.16 are not re-asserted here — no measured k_cat/K_m on this page [schematic]. Pair with a kinetics chapter.Source: BRENDA EC 2.3.1.16 — no k_cat/K_m re-asserted
Sequence Human ACAA2 UniProt P42765 · NCBI Gene 10449. This screen uses 4C2J numbering (Cys92 = UniProt Cys92). Chain A. Cα 1–396 with gap 210–213. His0 expression tag not this mesh.
Simulation map — what you see is what is written Setup = 4C2J pocket empty (opaque, no autoplay). ① schematic acetoacetyl moves in. ② slow-mo: blue Cys92-S → C3; fade to acetyl-CoA + acetyl-Cys. ③ first acetyl-CoA leaves. ④ crystal CoA moves in. ⑤ slow-mo: CoA-S → acetyl-Cys; fade to second acetyl-CoA. ⑥ second acetyl-CoA leaves. Only these events move.
Sources — bibliography of this page S1 — PDB 4C2J: human ACAA2 + CoA, 2.00 Å X-ray — public coordinates; measured Cys92 SG⋯CoA S1P = 3.98 Å · Cys92 SG⋯His352 NE2 = 3.24 Å · Cys92 SG⋯Cys382 SG = 5.79 Å S2 — Kiema et al., 2014, PMID:25478839 — human mitochondrial 3-ketoacyl-CoA thiolase with CoA S3 — Sequence: UniProt P42765 · NCBI Gene 10449 S4 — Kinetics: BRENDA EC 2.3.1.16 — no k_cat/K_m re-asserted on this page

› Backbones and CoA poses are taken directly from the listed PDB entries, superposed into one common frame. Where no crystalline state exists for a step (e.g., acetoacetyl-CoA on 4C2J, acetyl-Cys92, nucleophile flight, product leave), the pose is drawn schematically and labeled as such in the narration. Occupancy is free CoA, not 3-keto. Cys92 is CSO, not acetyl-Cys. Free-CoA Å are not 3-keto geomClaims.

N-domainC-domainONnucleophile (blue)