Succinyl-CoA Synthetase - 3D Mechanism Simulator

START HERE — how to read the succinyl-CoA → succinate + GTP picture SCS does substrate-level phosphorylation in three textbook moves: Pi splits the succinyl-CoA thioester (succinyl phosphate, CoA-SH leaves), His259α ★ takes the phosphoryl (succinate leaves), and the phosphohistidine loop swings 29 Å to hand the phosphoryl to GDP → GTP. The rare luck of this page: the crystal 2FP4 caught the phospho-enzyme WITH its GTP product — His259 phosphorylated on Nε (deposited NEP) and GTP in the β pocket — so both ends of the phosphoryl's journey are measured seats. Three honest asterisks travel with every number: the succinyl-CoA/GDP poses are schematic (no co-crystal), a second deposited Mg-GTP is crystal packing (not drawn), and this page's old His299/EC-6.2.1.5 labels were wrong and are retired here (A299 is a glutamate; the GTP enzyme is EC 6.2.1.4).Reading path (drawer-first, 5 minutes): 1 · Crystal structure — which seats are measured (2FP4 · GTP · K⁺ · 2.08 Å; packing Mg-GTP not drawn) · 2 · Mechanism — the three phosphoryl flights and the site I↔II swing (29 Å figure, 31.79 Å bound) · 3 · Step cards 0–④ — each beat and its number (2.58 / 2.91 / 2.44 / 2.84 / 3.00 pocket + settled NE2–P 1.74 + staged 5.93 gap) · 4 · Legend & Fidelity + Words used on screen — white-vs-gold arrow grammar and what “[schematic]” means · 5 · Simulation map — what the screen plays vs. what the code holds · then Sources — bibliography of every claim.
Crystal structure PDB 2FP4 — pig GTP-specific SCS αβ dimer with NEP259 (phospho-His) + GTP + K⁺, 2.08 Å, Fraser ME et al., J Biol Chem 2006, PMID:16481318. GTP sits in the ATP-grasp domain of the β subunit: the guanine base is recognized by Gln-20β (NE2⋯O6 2.58 Å) and the γ-phosphate is clamped by Arg-54β (NH2⋯O1G 2.91 Å). Companion frames (named, not drawn): dephospho/phospho pair 1EUC/1EUD (Fraser 2000) and the loop-at-site-II GDP complex 7JFP (Huang & Fraser 2021) and the CoA complex 4XX0 (Huang et al. 2015, PMID:26249701 — the S1P seat superposed for the ① dock).
Mechanism — His259α phosphohistidine + two sites His259α ★ (Nε-phosphorylated, N3-phosphohistidine) is the covalent intermediate; the phosphohistidine loop (α 248–270) swings it between site I (CoA/succinate/Pi chemistry) and site II (nucleotide pocket), ~29 Å apart per the 7JFP/2FP4 superposition — 31.79 Å P⋯γ-P inside 2FP4 itself. Partial reactions: (1) succinyl-CoA + Pi → enzyme-bound succinyl phosphate + CoA-SH (direct evidence: Nishimura & Meister 1965); (2) succinyl-P + His → succinate + His-P; (3) His-P + GDP → GTP. This page replaces the old His299 star: chain-A 299 is a glutamate in the deposited structure, and the phosphoryl sits on His259.Source: Fraser et al. 2006 (PMID:16481318) — frame + pocket · Huang & Fraser 2021 (7JFP) — loop at site II, 29 Å swing · Nishimura & Meister 1965 (succinyl-P evidence) · SEQADV NEP A259 (deposited phosphohistidine)
Phosphoryl transfers — what flies, and why no TS rig Three phosphoryl flights are drawn: (1) the PO3 visibly leaves Pi for the thioester carbonyl in ① — docked first, then flies, never mid-flight; (2) the phosphoryl is born in place at the transfer seat in ②, then settles onto His (short glide = loop-closure proxy) ending as the deposited His-P look; (3) it flies His → the vacant γ seat on GDP in ③ along the labeled loop road, completing the GTP pose atom-for-atom. Mechanism names, not repo slang: step ① is associative attack at carbonyl carbon — SN2(P)-type inline transfer does not apply (no pentacoordinate phosphorus, hence no equatorial rig). Steps ②–③ are formal SN2(P)-type displacements at phosphorus (inline geometry inferred from the seats, not measured — the loop carrying the phosphoryl is text plus the dashed arc, whose curve is staged).Source: partial reactions (1)–(3), see Mechanism card · inline-vs-carbonyl distinction is standard physical-organic nomenclature
0 — Setup: 2FP4 phospho-product The deposited enzyme holds phospho-His259 + GTP + K⁺ exactly as drawn — opaque, run:null. The residue mesh shows the His skeleton; its phosphoryl replays as the PHP marker from step ②. The second deposited Mg-GTP (B404) is crystal packing (no protein atom within contact range) and is not drawn. In-crystal P⋯γ-P = 31.79 Å: the two-site separation, claimed as a bound, never a bond.Source: PDB 2FP4 (2.08 Å), Fraser 2006
① — Phosphorolysis → succinyl-P + CoA-SH [schematic chemistry with one measured seat] Succinyl-CoA docks sulfur-first — its S sits on the 4XX0 CoA-S1P seat superposed onto this frame (302 Cα, RMSD 0.59 Å, seam disclosed; the free thiol reaches toward His in that complex) — and Pi beside it; the PO3 visibly flies Pi → carbonyl (associative attack at carbonyl C — SN2(P)-type inline transfer does not apply here, so no pentacoordinate P and no equatorial rig). Enzyme-bound succinyl phosphate forms; CoA-SH leaves. The 4XX0 phosphate at the His site is crystallization buffer, not a substrate pose — Pi's seat stays schematic.Source: partial reaction (1); S seat: Huang et al. 2015 (PMID:26249701, PDB 4XX0); succinyl-P as enzyme-bound intermediate per Nishimura & Meister 1965
② — His phosphorylation → succinate + His-P ★ His259α ★ takes the phosphoryl where it sits — docked near His, received there (staged attack gap 5.93 Å, honesty-bound; the mobile loop closes it in reality, static protein here). Succinate leaves; the phosphoryl settles onto His (short glide, loop-closure proxy) so the end state reads as His-P — settled NE2–P 1.74 Å beat. The transfer itself is a steep canonical cross-fade (no lingering ghost). Slow-motion on purpose.Source: partial reaction (2); deposited NEP259 (SEQADV) · Nε phosphorylation per Hultquist 1966
③ — GDP → GTP (five measured contacts) GDP waits in the β pocket; the phosphoryl flies in from His259 (schematic path between two deposited seats) and GTP swaps in over the same pocket. Five contacts label in turn, all re-measured from 2FP4: 2.58 / 2.91 / 2.44 / 2.84 / 3.00 Å, each single then cleared.Source: partial reaction (3); pocket geometry PDB 2FP4 (see tools/manifests/scs.json geomClaims)
④ — Products released — His + K⁺ stay home [schematic exits] GTP toward the GTP-driven reactions, succinate toward SDH, CoA-SH recycling — each on its own vector (Rule 4). His259 stays home for the next turn; K⁺ stays in the pocket. Net: succinyl-CoA + GDP + Pi → succinate + GTP + CoA-SH.Source: Rule 4 display convention — separate product exits; no external geometry claimed

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: the 2FP4 phospho-product frame (2.08 Å) with NEP259 + GTP-B403 + K401 as deposited seats, the five pocket contacts reproduced from 2FP4 into the manifest geomClaims (2.58 / 2.91 / 2.44 / 2.84 / 3.00 Å) plus the settled Nε–P bond (1.74 Å, sixth claim), the staged attack gap (5.93 Å, honesty-bound), and the 31.79 Å P⋯γ-P site separation as an honesty bound. The inferred layer (all [schematic]): every substrate/intermediate pose past the product frame — succinyl-CoA, Pi, succinyl-P, succinate, GDP seats, the phosphoryl flight path, the loop swing, and the product exits (step ④ is tagged [schematic] outright). The second Mg-GTP is deposited packing, excluded rather than disguised. Where a transition cannot be attached to a crystalline state, the drawerNote carries the schematic framing.
Why is the chemistry “[schematic]”? 2FP4 is a phospho-product complex: phospho-His + GTP, loop parked at site I. It shows both ends of the phosphoryl's journey but no substrate, no GDP, and no mid-swing loop — so the succinyl-CoA/GDP poses, the flight path, and the swing are rebuilt on/near the deposited seats and tagged [schematic], with the 31.79 Å separation stated instead of a fabricated trajectory. Everything drawn at the seats themselves (His skeleton, GTP, K⁺, phosphoryl marker, five contacts) travels on measured coordinates, which is why this page's schematic tag starts at the substrates, not at the pocket.
Catalytic residues — His259α ★ · Gln20β · Arg54β · Lys222β · Asp220β (+ K⁺) His259α ★ = covalent phospho-site (Nε, deposited NEP; the old His299 star pointed at a glutamate and is retired). Gln20β = guanine recognition (NE2⋯O6 2.58 Å). Arg54β = γ-phosphate clamp (NH2⋯O1G 2.91 Å). Lys222β = γ-phosphate contact (NZ⋯O1G 2.44 Å). Asp220β = K⁺ ligand (OD2⋯K 2.84 Å); K⁺ bridges the β-phosphate (3.00 Å). Retired pins (555/563/567/606/609/613) sit well clear of the deposited chemistry with no assigned role. Active site spans both subunits: the nucleotide pocket is β, the phospho-His is α.
Disagreements on record (a) Succinyl-P lifetime. Direct evidence as an enzyme-bound intermediate (Nishimura 1965, E. coli) — but a 2021 attempt to trap it on pig GTP-SCS recovered hydrolyzed succinate + phosphate instead (half-life ~39 min at pH 7.8 free); step ① is lit.-based. (b) Kinetic mechanism. Ordered ter-ter with dead-end succinate inhibition best fits the pig-heart data (PMC3549575) — older ping-pong/partially-random proposals are recorded there, not drawn. (c) EC number. This GTP enzyme is EC 6.2.1.4 (GDP-forming); the old page's 6.2.1.5 belongs to the ADP-forming isoform — corrected with BRENDA + pig-heart literature. (d) Loop swing path. The 29 Å figure is a 7JFP/2FP4 superposition interpolation; no trajectory is drawn.
Words used on screen — substrate-level phosphorylation · phosphohistidine · site I/II Substrate-level phosphorylation — ATP/GTP made by direct phosphoryl transfer from a high-energy substrate intermediate, not by a proton gradient; SCS is the TCA cycle's only one. Phosphohistidine — a histidine carrying a phosphoryl on a ring nitrogen (here Nε, the τ nitrogen); acid-labile, which is why it rarely survives crystallography — 2FP4 is the lucky exception. Site I / site II — the CoA/succinate/Pi chemistry site and the nucleotide site, 29 Å apart in one αβ dimer, linked by the swinging phosphohistidine loop. White arrow = direction of nucleophilic attack, tail at the attacker and head at the target — always local to the reaction site, never a transfer path. The flying group itself shows the transfer. Who attacks: ① Pi-oxygen → SCoA carbonyl-C · ② His-NE2 (nitrogen) → succinyl-P phosphorus · ③ GDP β-phosphate oxygen → incoming phosphorus. Never H+ — this page draws no proton chemistry; white arrows (not gold) mark these attacks so they never read as protons.
Kinetics scope (no measured timecourse on this page) One succinyl-CoA → GTP event is drawn, not a steady-state run. Pig-heart SCS: Km(GTP) ≈ 27–48 µM, kcat ≈ 65 s⁻¹ (Nishimura 1985/1988, attributed — see Sources); enzyme phosphorylation by GTP is near-equilibrium (ΔG° ≈ −400 cal/mol, Brownie & Bridger 1972). Best-fit kinetic model: ordered ter-ter with dead-end succinate inhibition against succinyl-CoA (PMC3549575 reanalysis of the Cha & Parks 1964 data).Source: Nishimura & Mitchell 1985 (Km/kcat) · Nishimura et al. 1988 (EC 6.2.1.4, dimer, Km) · PMC3549575 (kinetic model) · Brownie & Bridger 1972 (ΔG°)
Regulation — isoforms, reversibility, branch points Mammals run two SCS isoforms on one shared α (SUCLG1): GTP-specific (β = SUCLG2, this page) and ATP-specific (β = SUCLA2, NCBI Gene 8803) — the β subunit decides the nucleotide; defects cause mtDNA-depletion encephalomyopathy. The reaction is fully reversible near equilibrium: mass action (substrate/product ratios) sets the direction, and succinate dead-end-inhibits against succinyl-CoA. Succinyl-CoA is a branch point beyond the TCA (heme biosynthesis, ketone-body metabolism), so this step also gates those paths. Tissue split: ATPSCS runs higher in heart/brain/muscle, GTPSCS in kidney/liver (Lambeth et al. 2004, via Huang et al. 2015).Source: Fraser 2006 (two mammalian isoforms) · PMC3549575 (reversibility + dead-end inhibition) · NCBI Gene 8803 (SUCLA2 disease link) · Lambeth 2004 tissue split (via Huang 2015)
Simulation map — what you see is what is written Setup = the 2FP4 phospho-product (His skeleton + GTP + K⁺, opaque, run:null) with the NEP/packing/EC asterisks. ① succinyl-CoA + Pi dock at site I (His pinned, pocket labels retired); PO3 visibly flies Pi→carbonyl after docking; CoA-SH exits as succinyl-P fades in. ② STAR slow-mo: white attack arrow His→substrate P across the staged gap; steep cross-fade transfers the phosphoryl in place, settles onto His (reads as His-P, 1.74 settled beat); succinate exits. ③ GDP waits (γ seat labeled empty); the phosphoryl flies along the labeled loop road (arc fades in, arrow paired with the flight); five pocket contacts label in turn (2.58 / 2.91 / 2.44 / 2.84 / 3.00 Å) and clear; GDP fades as GTP (same pocket) fades in — atom-aligned seats, so the join reads as one molecule. ④ GTP exits alone (succinate left in ②, CoA-SH in ① — nothing exits twice); His + K⁺ stay. Only these events move; everything else is a static frame of 2FP4.
Sequence Pig α UniProt O19069 (SUCA_PIG; deposited 3..306 ↔ UniProt 29..332, His259 = UniProt His285) · pig β-GTP UniProt P53590 (SUCB2_PIG; deposited 2..395 ↔ UniProt 40..433) · human α P53597 · human β-GTP Q96I99 · human β-ATP (SUCLA2) Gene 8803. Screen shows 2FP4 deposited numbering throughout.
Sources — bibliography of this page S1 — PDB 2FP4 (2.08 Å): pig GTP-SCS αβ + NEP259 + GTP + K⁺; Fraser et al. 2006, J Biol Chem 281:11058–11065, PMID:16481318 S2 — PDB 1EUC/1EUD: phospho/dephospho pig GTP-SCS; Fraser et al. 2000, J Mol Biol 299:1325–1339 (loop α 248–270) S3 — PDB 7JFP: loop at site II (GDP complex); Huang & Fraser 2021, PMC7919408 (29 Å swing) S4 — Nishimura & Meister 1965, Biochemistry 4:1457–1462, DOI 10.1021/bi00883a034 (enzyme-bound succinyl-P evidence) S5 — Cha & Parks 1964, JBC 239:1961–1967, PMID:14213384 (purification) + 239:1968–1977 (kinetics data reanalyzed in S6) S6 — PMC3549575 (ordered ter-ter + dead-end succinate inhibition, pig heart) S7 — Nishimura & Mitchell 1985, JBC (Km(GTP) 48 µM, kcat 65 s⁻¹) · Nishimura et al. 1988, Biochem J 250:429 (EC 6.2.1.4, αβ dimer, Km 27 µM) · Brownie & Bridger 1972, Can J Biochem 50:719–724 (1 phospho-His/enzyme, ΔG° ≈ −400 cal/mol) S8 — UniProt P53597 (α) · Q96I99 (β-GTP) · Gene 8803 (SUCLA2) · BRENDA EC 6.2.1.4 · Repo display conventions: Rule 4 (separate product exits) · Rule 34 geometry (6 geomClaims reproduced in tools/manifests/scs.json) · [schematic]/same-seat swap = stated on screen, no deposition claimed S9 — PDB 4XX0: pig GTP-SCS + CoA + phosphate; Huang et al. 2015, Acta Cryst F 71:1067–1071, PMID:26249701 (CoA-S1P seat superposition source; crystallization phosphate at the His site is buffer, not a substrate pose)

› 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., substrate poses, the loop swing, product exits), the pose is drawn schematically and labeled as such in the narration.