Citrate Synthase - 3D Mechanism Simulator

Loading structural data… (5CTS)

References & Databases

START HERE — why things pop into existence If step ⑥ startled you, read this. Nothing flies in, because nothing arrives. Citryl-CoA is synthesized in place from the oxaloacetate and acetyl group already seated in the pocket — the same atoms, one new C–C bond. A delivery flight would teach false chemistry: products appear where their substrates were. In step ⑤ follow the solid bond stick — it lands first and never blinks; the small citryl head forms around it. In step ⑥ that same head gradually gains its tail context and becomes the full crystal analog (carboxymethyl-CoA): the long pale tail is only the CoA anchor, the chemistry already happened at the head. Nothing new ever enters.
Crystal structure PDB 5CTS — chicken citrate synthase closed ternary with oxaloacetate + carboxymethyl-CoA (citryl-CoA analog), 1.9 Å, Karpusas M, Branchaud B, Remington SJ, Biochemistry 29:2213-2219, 1990, PMID:2337600. D- and L-malate ternary complexes define the substrate position (Karpusas 1991, PMID:2043640).
Mechanism — ordered binding + enolization Oxaloacetate binds first and raises acetyl-CoA affinity ≈20×; open form admits substrates, closed form catalyzes (Wiegand & Remington, Annu Rev Biophys 15:97-117, 1986, DOI 10.1146/annurev.bb.15.060186.000525). Enolization is rate-limiting with inversion at the methyl carbon; citryl-CoA goes forward or back (Eggerer, via Remington review). Carboxymethyl-CoA is an enolization TS analog (Ki 0.08 µM).
Setup — 5CTS closed ternary crystal What you see is the deposited closed enzyme holding oxaloacetate and carboxymethyl-CoA (citryl-CoA analog). Open form admits substrates and releases products; closed form catalyzes.Source: PDB 5CTS (Karpusas et al. 1990, Biochemistry 29:2213-2219) — deposited closed ternary, OAA + carboxymethyl-CoA
① — OAA docks first Oxaloacetate binds first (ordered mechanism) into the loosened pocket. Arginines grip it — Arg401 salt bridge measured 2.89 Å in 5CTS. OAA seating raises acetyl-CoA affinity ≈20×; only the si-face stays open to attack.Source: Wiegand & Remington 1986 (ordered binding, ≈20× affinity) · Arg401–OAA O4 = 2.89 Å, measured in PDB 5CTS (Karpusas 1990)
② — Domain closes The displayed sticks glide slightly inward (schematic, at most 1.2 Å): open → closed around the seated OAA. It stays closed for the rest of the cycle.Source: Wiegand & Remington 1986 (open ↔ closed states) · ≤1.2 Å = registered schematic, not measured (Sources card below)
③ — Acetyl-CoA docks Acetyl-CoA docks second (schematic pose): methyl carbon ≈3 Å from the Asp375 base, thioester carbonyl facing His274 — the geometry the concerted step needs.Source: schematic pose — no CS·acetyl-CoA Michaelis crystal deposited; ≈3 Å registered (Sources card below); Asp375/His274 roles: Kurz et al. 1998
④ — Enolization (proposed) Concerted general acid–base (Karpusas 1990): Asp375 takes the methyl proton, His274 protonates the carbonyl oxygen — the gold balls are the two protons, the parked ball is the enol OH. Rate-limiting (Eggerer). M-CSA annotates an enolate instead.Source: Karpusas 1990 (concerted general acid–base, PDB 5CTS) · rate-limiting enolization per Eggerer (see Mechanism card) · enolate alternative: M-CSA entry 78
⑤ — Condensation to citryl-CoA Enol carbon attacks the OAA C2 carbonyl from the si-face (methyl stereochemistry inverts). The solid bond stick never blinks — OAA + acetyl, same atoms, one new C–C bond: citryl-CoA.Source: Wiegand & Remington 1986 (si-face attack, methyl inversion) · citryl-CoA forward/back per Eggerer (see Mechanism card)
⑥ — Citryl-CoA intermediate The small head gradually gains its context and becomes the full crystal analog, ghosted. The long pale tail is only the CoA anchor to the distant tunnel. Tightly bound: forward to hydrolysis or back to reactants (Eggerer).Source: PDB 5CTS (full analog = carboxymethyl-CoA) · forward/back per Eggerer (see Mechanism card)
⑦ — Hydrolysis (proposed) Water arrives, then splits: gold H⁺ to the CoA sulfur, OH⁻ to the thioester carbon (blue = lone pair). The C–S stick breaks; citrate + CoA-SH fade in. The Asp375-anhydride route is the recorded alternative.Source: proposal — direct water attack, no TS observed · recorded Asp375-anhydride alternative (M-CSA entry 78, see Disagreements card)
⑧ — Citrate + CoA-SH leave Products leave on different trajectories (Rule 4): citrate toward aconitase, CoA-SH recycled. Net OAA + acetyl-CoA + H₂O → citrate + CoA-SH, effectively irreversible in vivo.Source: Rule 4 — display convention, separate exits (Sources card below; no external geometry claimed)

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: the 5CTS chicken CS closed ternary (1.9 Å) and its D/L-malate companions as deposited OAA + carboxymethyl-CoA heavy atoms, plus the five mechanism contacts reproduced from those coordinates into the manifest geomClaims — Asp375–CMCoA methyl 3.32 Å (deprotonation seat), Arg401–OAA salt bridge 2.89 Å, OAA C2–analog C2 attack 3.70 Å, His274–analog O21 4.70 Å (suboptimal vs an ideal enol H-bond — the analog is not the true enol), His320–OAA carbonyl 2.90 Å (the Kurz polarizing contact). The inferred layer (all [schematic]): the ≤1.2 Å open→closed glide around seated OAA — registered, not measured; the docked acetyl-CoA ≈3 Å pose, because no CS·acetyl-CoA Michaelis complex is deposited; the neutral-enol concerted path, drawn as a proposal where M-CSA annotates an enolate; and the direct-water hydrolysis, whose TS has never been observed. Where a step is stitched between crystalline states the drawerNote carries the schematic framing.
Why is the acetyl-CoA pose "[schematic]"? No enzyme·acetyl-CoA Michaelis complex is deposited — 5CTS traps oxaloacetate + carboxymethyl-CoA, an enolization-TS analog, with the real acetyl group absent. The drawn acetyl pose therefore corrects carboxymethyl chemistry onto the trapped seats (methyl ≈3 Å off Asp375, thioester carbonyl toward His274, per Kurz 1998 role assignments) and carries the [schematic] tag. What is measured is the OAA + analog scaffold and the Asp375/His274/His320 contacts; what is rebuilt around it — the acetyl approach, the open-state glide, the hydrolysis water — is a literature-guided reconstruction, never a deposited geometry.
Catalytic residues — Asp375 · His274 · His320 Asp375 = base abstracting the acetyl-CoA methyl proton (also acts in hydrolysis); His274 = H-bond donor stabilizing the acetyl-CoA carbonyl in enolization and hydrolysis; His320 = polarizer of the OAA C2 carbonyl — NE2…the carbonyl O = 2.90 Å in the deposited 5CTS complex (Kurz et al., Biochemistry 1998, DOI 10.1021/bi980325g; M-CSA entry 78, ebi.ac.uk/thornton-srv/m-csa/entry/78).
Disagreements on record (a) Enol vs enolate — the animation draws the Karpusas neutral-enol concerted path as proposal; M-CSA annotates an enolate intermediate instead. (b) Hydrolysis — drawn as direct water attack (proposal); the Asp375-anhydride route (base: His320) is the recorded alternative. Neither TS has been observed.
Words used on screen — si-face · inversion · neutral enol si-face / re face — the flat OAA carbonyl can be attacked from either geometric face; only the si face is open in the pocket, the re face is blocked by the protein, so the condensation is stereospecific. Inversion at the methyl carbon — during enolization the two methyl protons change geometry (Eggerer), which is why CS builds a defined citrate carbon. Neutral enol vs enolate — the drawn path protonates the acetyl carbonyl oxygen with His274 (neutral enol, Karpusas 1990); M-CSA’s alternative keeps the carbanion deprotonated (enolate). And “OAA C2” in the narration = the C2 carbonyl carbon that the enol attacks — the His320-polarized carbonyl drawn on the crystal seat.
Kinetics (pig heart, wild-type) Km(acetyl-CoA) 0.005 mM · Km(OAA) 0.0059 mM · kcat 167 s⁻¹ (BRENDA EC 2.3.3.1, r488032, Kurz 1995); classic 17,000 mol citrate · min⁻¹ · (mol enzyme)⁻¹ = dimer turnover (Singh 1970, PMID:5466066) ≈ 140 s⁻¹ per subunit — in line with the BRENDA subunit kcat 167 s⁻¹. Dimer, MW 96,000.
Regulation Inhibited by ATP · NADH · succinyl-CoA (Smith & Williamson, FEBS Lett 1971, DOI 10.1016/0014-5793(71)80400-3) — the TCA gate closes when energy charge is high. Mechanism differs by source: for eukaryotic CS, ATP is a competitive inhibitor at the acetyl-CoA site (isosteric, not allosteric), while NADH is the clear allosteric inhibitor chiefly of Gram-negative bacterial CS (Harford & Weitzman, Biochem J 1975, 151:455-458).
Simulation map — what you see is what is written Setup = the 5CTS closed crystal. ① pocket loosened OPEN for entry; OAA move-in + Arg grip distance (label hides after its beat). ② the displayed catalytic sticks glide slightly inward around seated OAA (pocket tightens open→closed, ≤1.2 Å; labels stay pinned at crystal anchors). ③ acetyl-CoA move-in + methyl–Asp375 distance. ④ two gold balls fly at once (methyl-H → Asp375, His274-H → carbonyl O) in slow motion; the parked ball on the oxygen IS the enol OH. ⑤ the blue attack arrow draws the path FIRST, then acetyl glides down it (gap shrinks live); a solid bond stick lands and never blinks; OAA + acetyl (with its OH proton) fade INTO a small citryl head over the last quarter — the full CoA tail never pops here. ⑥ gradual crossfade across the whole step: the head becomes the ghosted crystal analog (pale tail = distant anchor). ⑦ water arrives at normal speed, then SPLITS in slow motion: gold H⁺ ball leaves for the CoA sulfur (water visibly loses it), the remainder is labeled OH⁻ nucleophile, glides onto the thioester carbon (blue = lone pair), the C–S stick vanishes (the break), citrate + CoA-SH (carrying the H) fade in. ⑧ separate exits. Every visible event above appears in its step narration; nothing else moves.
Sequence Chicken CS UniProt P23007 (frame: 5CTS author numbering — Asp375/His274/His320 as cited) · human mitochondrial CS UniProt O75390 · NCBI Gene 1431 (human CS)
Sources — bibliography of this page S1 — PDB 5CTS (1.9 Å): chicken CS closed ternary, OAA + carboxymethyl-CoA; Karpusas et al. 1990, Biochemistry 29:2213-2219, PMID:2337600; D/L-malate ternaries Karpusas et al. 1991, PMID:2043640 S2 — Wiegand & Remington 1986, Annu Rev Biophys Biophys Chem 15:97-117, DOI 10.1146/annurev.bb.15.060186.000525 (ordered binding, OAA-first preference and open ↔ closed forms; affinity figure quoted on the Mechanism card) S3 — Kurz et al. 1998, Biochemistry, DOI 10.1021/bi980325g (residue roles Asp375/His274/His320) S4 — M-CSA entry 78 (enolate alternative; curated mechanism) S5 — BRENDA EC 2.3.3.1, r488032 (Km/kcat values quoted on the Kinetics card); Singh et al. 1970, PMID:5466066 S6 — Smith & Williamson 1971, FEBS Lett, DOI 10.1016/0014-5793(71)80400-3 (ATP·NADH·succinyl-CoA inhibition) S7 — UniProt P23007 (chicken CS) · O75390 (human CS) · NCBI Gene 1431 S8 — Repo display conventions: Rule 4 (separate product exits) · registered schematics (≤1.2 Å closure, ≈3 Å dock) machine-verified in tools/manifests/cs.json (geomClaims + narrativeNumbers)

› 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., acetyl-CoA approach, water attack), the pose is drawn schematically and labeled as such in the narration.

Large domainSmall domainONS/P