ATP-citrate lyase - 3D Mechanism Simulator

START HERE — how to read citrate + ATP + CoA → acetyl-CoA + OAA + ADP + Pi A citrate-cleavage cartoon on the human ACLY frame that actually exists: 6UUW (2.85 Å EM) E599Q chain A with crystal ADP + CoA + Y2A (citryl-phosphate analog). Occupancy is ADP + CoA + Y2A, not ATP, not citrate, not acetyl-CoA — never copy the PDB title “Mg, citrate, ATP” as occupancy. There is no MG entity. UNL is named, not a substrate. Residue 599 is GLN (E599Q) — Glu599 is not this mesh. His760 ★ is the UniProt tele-phosphohistidine (phosphate ATP → citrate) — not a citryl carrier; no citryl-His has ever been observed. ATP / free citrate / acetyl-CoA / OAA / Pi are [schematic]. ATP and citrate bind the same ASH active site; the ~36 Å here is the ATP-grasp/citryl sub-pocket separation in this open, D2-symmetric mesh, not two domains — not a geomClaim. White is phosphoryl (PO₃ from ATP PG onto citrate-OH via His760), not nucleotidyl. Blue is CoA-S onto citryl-phosphate, not hydride. ACC1 next. Measured Å are deposited 6UUW analog pairs only.
Crystal / EM structures PDB 6UUW — human ACLY E599Q + ADP + CoA + Y2A, 2.85 Å cryo-EM, EMD-20902 (Wei 2020). Coordinates are public. Occupancy is ADP + CoA + Y2A. Human ACLY UniProt P53396.
Mechanism — phosphohistidine, then citryl-CoA; cleavage module contested UniProt P53396 EC 2.3.3.8 writes the reverse (citrate synthase) direction. This page teaches citrate cleavage for FAS: (1) citrate + ATP → citryl-P + ADP (His760 phosphohistidine). (2) citryl-P + CoA → citryl-CoA + Pi. (3) citryl-CoA → acetyl-CoA + OAA (retro-aldol cleavage — module contested: Wei 2020 ASH vs Verstraete 2021 CSH). Wei et al., Nat. Struct. Mol. Biol. 2020, PMID:31873304. ACC2 is another gene. Source: UniProt P53396 · Wei 2020
0 — Setup: empty citrate-cleavage pocket 6UUW occupancy is ADP + CoA + Y2A (citryl-P analog), not ATP, not citrate, not acetyl-CoA. The PDB title says Mg / citrate / ATP — that is not occupancy (no MG entity). Residue 599 is GLN (E599Q). The pocket is shown empty so binding can start. Opaque, run:null (no autoplay). His760 ★ is the tele-phosphohistidine. No citryl-His exists — the intermediate is citryl-phosphate/citryl-CoA (Y2A analog; exact 6UUW identity disputed). UNL is named, not a substrate. Cα gaps 140–148 / 432–486.Source: PDB 6UUW (2.85 Å EM)
① — Citrate binds [schematic] Citrate docks on the 6UUW Y2A pose — not deposited citrate. Y2A is a citryl-phosphate analog, not free citrate and not acetyl-CoA. E599Q: residue 599 is GLN. Product-analog Å are not citrate geomClaims.Source: 6UUW Y2A pose rebuilt as citrate
② — ATP binds Schematic ATP docks as crystal ADP plus a teaching γ-phosphate on the ADP pose — [schematic], never label crystal ADP as ATP. ATP and citrate occupy the same ASH active site — the ~36 Å here is the ATP-grasp/citryl sub-pocket separation in this open D2-symmetric mesh, not two separate sites. ATP binding compacts the ASH domain; the His760 loop (752-767) flaps between the two sub-pockets to deliver the γ-P. Domain closure is not animated. Lys58 NZ ⋯ ADP O2A = 3.18 Å and Asp216 OD2 ⋯ ADP O2A = 3.22 Å are both deposited 6UUW, not ATP geomClaims. Lys58 and Asp216 sit in this ATP sub-pocket, so they are shown here rather than on the citryl site. No crystal Mg.Source: S1 6UUW ADP pose rebuilt as ATP · S7 ATP + citrate bind the same ASH domain (PMC10115795) · S5 ASH compaction on ATP binding (PMC8436250)
\u2462 — His760 relay: ATP \u03b3-P \u2192 His760-P \u2192 citrate-OH (one ASH active site) (hypothetical, lit.-based) The real PO₃ atomic group (PG+O1G/O2G/O3G detached from ATP - real PO3 group + white arrow, never a ball) makes two hops, not one: an equatorial rig rides the travelling P with a 180-degree tick sweep, reading in-line attack with SN2 inversion (umbrella flip) at each hop. Beat 1 (ATP-pocket close-up): ATP PG gives its PO3 by in-line SN2(P), with ADP released; the PO3 travels the full PG-His760 axis to dock on His760 NE2 and dwells there visibly - phosphohistidine aboard the loop - while the camera glides over. Beat 2 (citryl-pocket close-up): His760-P sits on NE2, then citrate OHB, the prochiral 2-hydroxyl, attacks it to give citryl-phosphate; the PO3 lands exactly on the deposited Y2A phosphate (3.68 A from NE2), so the product fade-in is seamless. Schematic CIT and deposited Y2A poses differ by ~6 A in this mesh - landing on the deposited site keeps continuity. That relay is measured, not bookkeeping: [γ-³³P]-ATP pre-steady-state phosphorylation shows a pKa ≈ 7.5 ionisable group, and [γ-¹⁸O₄]-ATP β,γ-bridge↔nonbridge positional isotope exchange runs at 14 s⁻¹ with no citrate present (Wei 2012 Biochem; Wei 2020 NSMB). A direct citrate-on-ATP attack would make no phosphorylated-enzyme intermediate and so no isotope exchange. SN2-type phosphoryl transfer - what that means here. Each hop is an in-line SN2(P) event: the nucleophile attacks the phosphorus from directly behind the leaving group (near 180 degrees), through a trigonal-bipyramidal transition state with entering and leaving oxygens at the two apices. The three equatorial oxygens invert like an umbrella - each hop inverts the configuration at P, two hops give net retention. That is why the transfer must read as a direct head-on arrival and never a curved throw: the white shaft runs the true donor-acceptor axis and the rig's 180-degree tick is the flip made visible. Citrate + ATP cross-fade in place to crystal Y2A (citryl-P analog) and crystal ADP. Never label ADP as ATP. Never label Y2A as citrate. His760 ★ is this relay, not a citryl carrier. Citrate OG2 is a carboxylate oxygen, not the nucleophile — the phosphorylated O is OHB. Both hops run inside the one ASH active site, after the ASH domain compacts - and each hop is drawn ONLY as its own local close-up of the scene right before the transfer. (1) What you see vs what happens. What you see is the open 6UUW mesh, where the ATP-grasp and citryl sub-pockets sit ~36 A apart; what happens in solution is ASH compaction, after which both transfers run at bond distances (~2 A) inside the one active site. The gap is a crystal-state fact, never the reaction distance. (2) The loop is the carrier. The 752-767 loop that physically carries the γ-P is not in this residue set and is not drawn, and what is drawn is the loop's net result: the PO3 travels continuously from PG to His760 along the true axis - nothing vanishes mid-shot and reappears elsewhere, so this is not teleport: one object, one path, two windows. Beat 1 shows the departure in the ATP pocket; the camera glide follows the journey; beat 2 shows the docking, a visible dwell on NE2, and the short hop onto the Y2A phosphate site. The length is forced: the open 6UUW mesh holds the sub-pockets ~36 A apart while the closed enzyme - which this page cannot draw for lack of closed-state coordinates - brings them together, so the journey axis has no choice but to be drawn long. The distance is a crystal-state fact, never the reaction distance. (3) Why nothing more is drawn. The protein mesh is static by rule, there are no closed-state coordinates for this trajectory, and no geometry is fabricated: the loop itself, the closed domain pose, and the journey between beats are carried by words and the camera glide - with the PO3's net path drawn as one continuous axis, none of it fabricated as measured structure. Beat 2 lands exactly on the deposited Y2A phosphate and the product fades in over the PO3 group — one continuous PO3, no jump and no leftover marker; relay legs show the PO3 group + thin shaft only. That 6UUW traps ADP together with the citryl-P analog — precisely the ADP-plus-phosphorylated-citrate co-existence a two-step relay predicts. Slow-motion on purpose. White = phosphoryl (PO₃), not nucleotidyl, not hydride. E599Q.Source: S5 6UUW = ADP + phospho-citryl-CoA (PMC8436250) · S6 His760 loop 752-767 flaps between the two pockets (PMC11365397) · S2 Wei 2012 Biochemistry + Wei 2020 NSMB (positional isotope exchange)
④ — CoA binds (hypothetical, lit.-based) Crystal CoA docks — real 6UUW occupancy, not acetyl-CoA. CoA S1P ⋯ Y2A OG1 = 3.05 Å is the deposited analog contact (citryl-phosphate analog pose). Thr348 OG1 ⋯ Y2A OHB = 2.43 Å is deposited. Pi not yet formed. E599Q. ADP stays in the ATP-grasp pocket.Source: UniProt P53396 His760 · 6UUW His760
⑤ — CoA-S attacks citryl-phosphate → citryl-CoA + Pi CoA thiol S (blue ball + blue arrow) attacks citryl-phosphate. Citryl-phosphate + CoA cross-fade in place to schematic citryl-CoA + schematic Pi, which then leaves. No citryl-His exists — His760 is the phosphohistidine of step ③, not a citryl carrier (Wei 2020 Author Correction: the 6UUW intermediate may be phospho-citrate + CoA, citryl-CoA + phosphate, or a mixture; E599's cleavage role is unknown). His760 NE2 ⋯ Y2A O4 = 2.82 Å and NE2 ⋯ P = 3.68 Å are deposited analog contacts. Residue 599 is GLN (E599Q). Slow-motion on purpose. Blue = sulfur nucleophile, not hydride.Source: PDB 6UUW CoA
⑥ — citryl-CoA → acetyl-CoA + OAA (cleavage, contested module) (hypothetical, lit.-based) Schematic citryl-CoA cross-fades in place to schematic acetyl-CoA and schematic oxaloacetate — retro-aldol cleavage. Where cleavage happens is contested: Wei 2020 (ASH domain) vs Verstraete 2021 (CSH module) — this page does not pick a module. E599's role is unknown per the Wei Author Correction. Slow-motion on purpose. blue ball + blue arrow marks the cleavage center, not hydride. E599Q.Source: compiled ACLY thiolysis · Wei 2020
⑦ — Acetyl-CoA and OAA leave — ACC1 next Acetyl-CoA and oxaloacetate depart in opposite directions. ADP stays in the ATP-grasp pocket (already formed in ③). Pi already left. Acetyl-CoA is the substrate of ACC1 (next). This page does not carboxylate malonyl-CoA. 6UUW is E599Q. Y2A is a citryl-P analog, not citrate. UniProt writes the reverse (citrate synthase) direction; this page teaches cleavage. Analog Å are not ATP/citrate geomClaims.Source: FAS bookkeeping · EC 2.3.3.8

How to read this page — a first-pass primer First pass: Setup (empty pocket, His760 ★, E599Q, occupancy is ADP+CoA+Y2A) → ① schematic citrate docks on Y2A pose → ② schematic ATP docks on ADP pose → ③ real PO₃, two close-ups: long PG-to-His760 journey with His dwell, then His-P to citrate OHB; fade to Y2A + ADP → ④ crystal CoA docks → ⑤ blue CoA-S → citryl-phosphate, fade to citryl-CoA + Pi leave → ⑥ citryl-CoA fades to acetyl-CoA + OAA (cleavage module contested) → ⑦ acetyl-CoA and OAA leave opposite; ADP stays; ACC1 next. Second pass: 2.82 / 3.68 / 3.05 / 2.43 / 3.18 Å are crystal facts on 6UUW analog occupancy; ATP, citrate, acetyl-CoA, OAA, Pi are [schematic]. The ~36 Å ATP-grasp/citryl sub-pocket gap is an open-mesh fact within one active site, not a geomClaim. Third pass: open “Disagreements” for E599Q vs WT, Y2A vs citrate, ADP vs ATP, title vs occupancy, and UniProt reverse vs FAS cleavage.
Chemistry primer — citryl-P, then citryl-CoA, then contested cleavage ACLY cleaves citrate for cytosolic acetyl-CoA. Phosphoryl (PO₃) does not fly straight from ATP γ-P onto citrate: it makes two hops through catalytic His760. ATP PG → His760 NE2 (in-line SN2(P)) gives a phosphohistidine (His760-P) and releases ADP; citrate's prochiral 2-hydroxyl OHB then attacks His760-P, giving citryl-phosphate. Both hops run inside the one ASH active site: the ~36 Å between the ATP-grasp and citryl sub-pockets in this open mesh closes by ASH compaction, and the 752-767 loop delivers the γ-P. The evidence is the pKa ≈ 7.5 ionisable group in [γ-³³P]-ATP pre-steady-state phosphorylation and the β,γ-bridge↔nonbridge positional isotope exchange of [γ-¹⁸O₄]-ATP (14 s⁻¹, no citrate) — both require a phosphorylated enzyme intermediate. Note the atom: citrate OG2 is a carboxylate O on CGC; the phosphorylated O is OHB on the central CB. CoA-SH then attacks citryl-phosphate; citryl-CoA forms and Pi leaves. Citryl-CoA cleaves to acetyl-CoA + oxaloacetate — the cleavage module is contested (ASH vs CSH), so this page does not stage it in either domain. White = phosphoryl (PO₃ from ATP PG onto citrate-OH via His760), not nucleotidyl. Blue = CoA-S onto citryl-phosphate, not hydride. This is not ACC1 (carboxylation) and not ACC2.
Words used on screen — E599Q · Y2A analog · ADP ≠ ATP · His760 ★ · one ASH active site E599Q — residue 599 is GLN on this mesh; Glu599 is not here. Y2A analog — citryl-phosphate analog, not free citrate and not acetyl-CoA. ADP ≠ ATP — crystal occupancy is ADP; teaching ATP is schematic on that pose. His760 ★ — UniProt tele-phosphohistidine (ATP → citrate phosphate transfer); no citryl-His exists. One ASH active site — ATP and citrate bind the same ASH domain; ~36 Å is the ATP-grasp/citryl sub-pocket separation in this open, D2-symmetric mesh, not a domain split, not a geomClaim.

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: 6UUW (2.85 Å EM) human ACLY E599Q + ADP + CoA + Y2A; His760 NE2 ⋯ Y2A O4 = 2.82 Å; His760 NE2 ⋯ Y2A P = 3.68 Å; CoA S1P ⋯ Y2A OG1 = 3.05 Å; Thr348 OG1 ⋯ Y2A OHB = 2.43 Å; Lys58 NZ ⋯ ADP O2A = 3.18 Å. The inferred layer ([schematic]): ATP γ-P, free citrate, citryl-CoA intermediate, Pi, acetyl-CoA, OAA, transferring nucleophile spheres, product leave. Binding order is compiled staging, not a kinetic fit. The protein mesh is static. Analog Å are not ATP/citrate geomClaims. The ~36 Å ATP-grasp/citryl sub-pocket gap is an open-mesh fact, not a geomClaim. Title “Mg, citrate, ATP” is not occupancy.
Disagreements on record (a) Occupancy vs title. 6UUW holds ADP + CoA + Y2A, not ATP, not citrate, not acetyl-CoA. The title names Mg/citrate/ATP — no MG entity. (b) E599Q vs WT. Residue 599 is GLN. Glu599 is not this mesh. (c) Y2A vs citrate vs acetyl-CoA. Y2A is a citryl-P analog. (d) No citryl-His. No citryl-His adduct has ever been observed — the intermediate is citryl-phosphate/citryl-CoA (Y2A analog; exact 6UUW identity disputed per the Wei Author Correction). (e) Concerted vs split phosphorylation / citryl-CoA formation / cleavage — this page splits ③ then ⑤ then ⑥ for 1-event/1-step teaching. (e2) Cleavage module contested: Wei 2020 (ASH domain) vs Verstraete 2021 (CSH module) — this page does not pick a module; E599's role is unknown. (f) UniProt writes the reverse (citrate synthase) direction; this page teaches cleavage. (g) UNL is named, not a substrate. (h) The ~36 Å ATP-grasp/citryl gap is sub-pocket separation within ONE ASH active site in an open D2-symmetric mesh — not two domains, not a geomClaim. (i) ACC2 is another gene. (j) Phosphorylation / sugar-P activation = Regulation, not motion. (k) Cα gaps 140–148 / 432–486. (l) Tetramer A–D; teaching = chain A.
Regulation — phosphorylation · sugar-P · tetramer This cleavage is cytosolic ACLY only. ACC2 is another gene — not this mesh. Phosphorylation and sugar-P activation are named here, not animated. Tetramer assembly is not this cartoon. Domain closure between ATP-grasp and citryl pockets is not animated.Source: UniProt P53396 · PDB 6UUW · Wei 2020
Kinetics scope (no measured timecourse on this page) One citrate + ATP + CoA → acetyl-CoA + OAA + ADP + Pi event is drawn, not a steady-state run. Compiled BRENDA values for EC 2.3.3.8 are not re-asserted here — no measured k_cat/K_m on this page [schematic]. Pair with a kinetics chapter.Source: BRENDA EC 2.3.3.8 — no k_cat/K_m re-asserted
Sequence Human ACLY UniProt P53396 · NCBI Gene 47. This screen uses 6UUW numbering (His760 = UniProt His760; residue 599 = GLN). Chain A. Cα 2–1099 with gaps 140–148 / 432–486.
Simulation map — what you see is what is written Setup = 6UUW pocket empty (opaque, no autoplay). ① schematic citrate moves in. ② schematic ATP moves in. ③ slow-mo: real PO₃, two close-ups — long PG-to-His760 journey with His dwell, camera glides along, citryl-pocket hop His760-P → citrate OHB; fade to Y2A + ADP. ④ crystal CoA moves in. ⑤ slow-mo: blue CoA-S → citryl-phosphate; fade to citryl-CoA + Pi leave. ⑥ slow-mo: citryl-CoA fades to acetyl-CoA + OAA (cleavage module contested). ⑦ acetyl-CoA and OAA leave opposite; ADP stays. Only these events move.
Sources — bibliography of this page S1 — PDB 6UUW: human ACLY E599Q + ADP + CoA + Y2A, 2.85 Å EM — public coordinates; measured His760 NE2⋯Y2A O4 = 2.82 Å · NE2⋯P = 3.68 Å · CoA S1P⋯Y2A OG1 = 3.05 Å · Thr348 OG1⋯Y2A OHB = 2.43 Å · Lys58 NZ⋯ADP O2A = 3.18 Å S2 — Wei et al., Nat. Struct. Mol. Biol. 2020, PMID:31873304 — human ACLY cryo-EM (phospho-His760; 6UUW intermediate) + Author Correction (Y2A identity disputed; E599 role unknown) + Verstraete et al. 2021 Matters Arising (cleavage in CSH module) + Wei et al. 2021 Reply — no citryl-His in any source S3 — Sequence: UniProt P53396 · NCBI Gene 47 S4 — Kinetics: BRENDA EC 2.3.3.8 — no k_cat/K_m re-asserted on this page S5 - Wei et al., Nature 2021 (Molecular Basis for Acetyl-CoA Production by ATP-Citrate Lyase), PMC8436250 - 6UUW (ACLY-E599Q) active-site density modelled as ADP (hydrolyzed ATP) + phospho-citryl-CoA, i.e. ADP present together with the phosphorylated-citrate species; ATP binding drives the ASH domains into a more compact position than apo; ASH = residues 1-806, CSH = 859-1101. Source for the one-ASH-active-site framing and for ADP in the end state. S6 - Shi et al., iScience 2024 (Catalytic mechanism study of ATP-citrate lyase during citryl-CoA synthesis process), PMC11365397 - His760 loop (752-767) flaps between the Mg²²-ATP pocket and the citrate pocket, mediating the phosphate transfer for reaction (i); PIX rate 14 s⁻1. Source for loop-delivered relay inside one active site, and for the loop not being drawn on this page. S7 - Allosteric role of the citrate synthase homology domain of ATP citrate lyase, PMC10115795 - citrate and ATP bind the ASH domain; the CSH module forms a central homotetramerization core and plays an allosteric (not separate-site catalytic) role. Source for ATP + citrate sharing one active site.

› Backbones and ADP / CoA / Y2A poses are taken directly from the listed PDB entries, superposed into one common frame. Where no crystalline state exists for a step (e.g., ATP, free citrate, citryl-CoA intermediate, Pi, acetyl-CoA, OAA, nucleophile flight, product leave), the pose is drawn schematically and labeled as such in the narration. Occupancy is ADP + CoA + Y2A analog, not ATP/citrate/acetyl-CoA. Analog Å are not ATP/citrate geomClaims. 6UUW is E599Q.