Triosephosphate Isomerase - 3D Mechanism Simulator

Loading structural data… (1NEY · 7TIM)

References & Databases

Michaelis complex — 1NEY, the 1.2 Å trapped state 1NEY = the DHAP Michaelis complex of yeast (S. cerevisiae) triosephosphate isomerase at 1.2 Å — a state normally so short-lived it is hard to trap; Jogl G, Rozovsky S, McDermott AE, Tong L, Proc Natl Acad Sci USA 100:50–55, 2003, RCSB 1NEY · PMID:12509510 · DOI:10.1073/pnas.0233793100. Note: the crystal is a W90Y/W157F/W168(5-F-Trp) variant whose kinetic parameters are essentially wild-type (see the paper); DHAP here is the deposited ligand resName 13P.
Transition-state analog — 7TIM, superposed onto 1NEY 7TIM = yeast TIM bound to phosphoglycolohydroxamate (PGH), a 2-carbon hydroxylamide that mimics the cis-enediolate transition state (Davenport RC, Bash PA, Seaton BA, Karplus M, Petsko GA, Ringe D, Biochemistry 30:5821–5826, 1991, RCSB 7TIM · PMID:2043623). On this page the 7TIM coordinates were superposed onto the 1NEY frame by a whole-Cα fit (246 Cα, RMSD 0.285 Å), so the PGH phosphate overlies the DHAP phosphate in the same seat (P↔P ≈ 0.22 Å).
① DHAP docked — the real 1NEY Michaelis pose DHAP is shown at its deposited 1NEY coordinates: Glu165★ carboxylate ⋯ C2 = 2.99 Å and ⋯ C3 (CH₂OH end) = 3.06 Å (both measured). Jogl et al. call the close carboxyl–ketone/α-hydroxy carbon contact (≈3.0 Å to both carbons) "unprecedented"; the initial pro-R proton transfer happens at the C3 (CH₂OH) end, while the 2.99 Å C2 contact is the center Glu165 later reprotonates. His95 ⋯ O2 = 2.72 Å, His95 ⋯ O3 = 2.97 Å, Lys12 ⋯ O2 = 3.07 Å (all measured here from the deposited atoms).Source: PDB 1NEY (Jogl et al. 2003, PNAS 100:50-55) — measured Michaelis distances 2.99/3.06/2.72/2.97/3.07 Å from the deposited atoms (manifest geomClaims)
② Enediolate / pro-R proton abstraction — PGH surrogate [schematic proton path] Glu165★ is the general base: it abstracts the pro-R proton from the CH₂OH (hydroxymethyl) end of DHAP — the carbon that becomes the aldehyde C1 of D-G3P (Jogl 2003; classic TIM literature). The labile ketone collapses to the cis-enediolate, which is never observed directly; the page represents it by the superposed PGH TS-analog cross-fading in place — its oxygens sit near His95 (2.67 Å), Lys12 (2.84 Å) and Glu165 (2.90 Å; measured). The gold H⁺ ball is a deliberately drawn cartoon (Rule 23), not a quantum H-transfer trajectory.Source: PGH surrogate, PDB 7TIM (Davenport et al. 1991) fitted onto 1NEY by whole-Cα fit (246 Cα, RMSD 0.285 Å) — PGH contacts 2.67/2.84/2.90 Å measured (manifest geomClaims) · enediolate = registered schematic, not measured · Glu165 single-base shuttle: M-CSA entry 324
③ Reprotonation → D-G3P; re-annotation disclaimer [schematic] Glu165–H reprotonates C2 and the carbonyl migrates from C2 to the former CH₂OH end — the net C2→C1 isomerization. D-G3P is drawn as the same skeleton re-annotated in place (the deposited 1NEY substrate is DHAP; no G3P complex is co-crystallized here), so the phosphate stays seated and the molecule is re-labeled: the aldehyde C1 appears at the old CH₂OH end, C2 becomes CH(OH). The motion between the three static states (1NEY → 7TIM → re-annotation) is stitched and schematic; in the enediolate the two substrate oxygens share the developing negative charge (His95 / Lys12 shepherd both).Source: re-annotation = display convention — 1NEY holds DHAP only, no TIM·G3P crystal co-deposited (PDB 1NEY) · inter-state motion = registered schematic, not measured · single-Glu165 shuttle per M-CSA entry 324

How to tell a proposal from a fact on screen Deposited geometry (no marker) = measured from the listed PDB coordinates: the 1NEY Michaelis distances (Glu165⋯C2 2.99 Å, Glu165⋯C3 3.06 Å, His95⋯O2 2.72 Å and ⋯O3 2.97 Å, Lys12⋯O2 3.07 Å) and the superposed PGH contacts (2.67/2.84/2.90 Å), plus the Cα-fit RMSD 0.285 Å. [schematic] = the proton ball, the arrow, and the inter-state cross-fades (② enediolate formation, ③ G3P re-annotation) — a transition state is never observed, so every arrow here is a literature-based proposal. The gold arrow marks the path traced by the cartoon ball — a trajectory marker, not a curly-arrow electron-pushing step; the real H-transfer is quantum-level (tunneling/zero-point), not a discrete ball hop.

Legend & Fidelity

Mechanism fidelity The page shows the classical single-Glu165 proton shuttle (Glu165 is the only base; the same carboxyl abstracts the pro-R proton and later reprotonates). The isomerization is reversible and symmetric through the same enediolate, but the equilibrium lies far toward DHAP: K_eq = [G3P]/[DHAP] ≈ 0.045 — i.e. ≈ 96% DHAP (DHAP:G3P ≈ 96:4) at equilibrium (Wierenga, Cell Mol Life Sci 67:3961–3982, 2010, PMID 20694739) — which is why net DHAP→G3P flux in glycolysis is mass-action driven: aldolase feeds DHAP in and GAPDH drains G3P out (see the regulation card). Partitioning of the enediolate is stereospecific: the closed loop 6 "lid" (residues 166–176; its tip ≈ 7 Å open↔closed in the yeast enzyme is a literature comparison between open and closed crystal structures — not measured on this page's single closed 1NEY snapshot) seals the pocket against water and pins the substrate, so reprotonation always lands on the same C2 face. The literature also weighs a "criss-cross" proton-routing hypothesis (diffusing proton vs. a second proton relay) — Glu165 remains the assigned base in the primary works; the shuttle below is the lit.-based main route, with the alternatives noted in M-CSA Entry 324.
Why is the intermediate shown as a surrogate "[schematic]"? Because the species that links DHAP and G3P is too short-lived to be trapped — no crystal holds it. The page therefore stands in the superposed 7TIM PGH analog (see the ② card) and cross-fades it in and out; the gold proton ball and that cross-fade are literature-based cartoons, not a measured species. The crystallographic anchors are the two endpoint states — the 1NEY pose and the 7TIM coordinates fitted onto it — with the fit reported in the reference cards above.
Precision vs. accuracy — Å numbers are single crystallographic states Distances shown are single values from the deposited entries (1NEY Michaelis) or our own common-frame superposition after the Cα fit (PGH contacts), not measured dynamics. The scene joins two structures (1NEY frame + 7TIM PGH overlay) into one frame; inter-state transitions and the proton path are stitched, schematic cartoons. Treat every Å as a plausible crystallographic value, not a measured coordinate of the moving state.
The protein mesh is static — the loop-6 "lid" is a closed snapshot, not dynamics The protein mesh is static: no conformer sampling, thermal motion, or loop dynamics are simulated. The loop-6 "lid" (residues 166–176; its tip ≈ 7 Å open↔closed in the yeast enzyme is a literature comparison between open and closed crystal structures — not a distance measured on this page's single closed snapshot) is therefore shown only as the closed crystallographic snapshot — its opening/closing and the induced-fit sealing against water are disclosed by the label and the narration, not animated. Animations interpolate between real crystallographic states (1NEY → 7TIM PGH overlay → re-annotated G3P), and any reconstruction is "literature-based", not measured.
Physiological regulation — a near-equilibrium step; mass action, not allostery TPI sits near equilibrium (its G3P↔DHAP couple is not a committed step; ΔG°′ is small and the enzyme is fast in both directions), and no allosteric regulator is recognized — none is drawn here. Direction is set by mass action: aldolase feeds DHAP in and GAPDH drains G3P during glycolysis; the flow reverses in gluconeogenesis. Human TPI is a single ubiquitous isozyme (TPI1, UniProt P60174) with no tissue-switched allosteric isoform of the PFK-1 kind — the "kinetically perfect" label refers to catalytic efficiency, not to flux control.
Kinetics scope — why "kinetically perfect" This tool animates a single catalytic event; it does not model timecourses, steady-state kinetics, or K_m / k_cat / V_max · [S] behavior. The label "kinetically perfect" comes from TPI being diffusion-limited in the direction whose substrate is scarce: for yeast TPI, GAP→DHAP has k_cat/K_m near the diffusion-encounter limit (maximum ≈ 10⁹ M⁻¹·s⁻¹; measured ≈ 3.7×10⁸ M⁻¹·s⁻¹ on the unhydrated aldehyde), while the physiological DHAP→G3P direction is not diffusion-limited — k_cat ≈ 500 s⁻¹, K_m(DHAP) = 1.2 mM (0.7 mM unhydrated), i.e. k_cat/K_m ≈ 4×10⁵ M⁻¹·s⁻¹ on total concentrations (free-energy analyses: Albery & Knowles, Biochemistry 15:5627, 1976, PMID 999838; Wierenga, Cell Mol Life Sci 67:3961–3982, 2010). Wild-type TIM second-order efficiency is diffusion-controlled, shown by viscosity variation (Blacklow et al., Biochemistry 27:1158, 1988, PMID 3365378; see also BRENDA EC 5.3.1.1 and the kinetic values compiled for M-CSA Entry 324). Anything faster than every encounter would be wasted — TPI is a textbook near-perfect catalyst.
Sequence Gene TPI1 (S. cerevisiae) · protein UniProt P00942. Residue numbers on screen follow the PDB-author numbering of the 1NEY/7TIM chain A sequences (S. cerevisiae, residues 2–248; both structures share the same numbering, so Glu165/His95/Lys12/Asn10/Glu97 correspond directly). Residue 168 of the loop-6 lid is the 5-fluorotryptophan (W168 → FTR) of this mutant's crystals — a HETATM, so it contributes no Cα to the on-screen mesh or the 246-Cα fit (the author numbering 2–248 still spans it).
Sources — bibliography of this page S1 — PDB 1NEY (1.2 Å): yeast TIM DHAP Michaelis complex (W90Y/W157F/W168-F-Trp variant, kinetically essentially wild-type); Jogl G, Rozovsky S, McDermott AE, Tong L, PNAS 100:50–55, 2003, PMID:12509510 · DOI 10.1073/pnas.0233793100 — measured Michaelis contacts (Glu165 / His95 / Lys12) as shown on the ① card, values registered in tools/manifests/tpi.json S2 — PDB 7TIM: yeast TIM bound to phosphoglycolohydroxamate (PGH), the cis-enediolate TS-analog; Davenport RC, Bash PA, Seaton BA, Karplus M, Petsko GA, Ringe D, Biochemistry 30:5821–5826, 1991, PMID:2043623 — superposed onto 1NEY by the whole-Cα fit (fit and PGH oxygen distances shown on the ② card; geomClaims in tools/manifests/tpi.json) S3 — M-CSA entry 324: classical single-Glu165 proton shuttle (main lit.-based route); criss-cross / second-relay alternatives noted there, not drawn S4 — Kinetics context: Albery WJ & Knowles JR, Biochemistry 15:5627, 1976, PMID:999838 (free-energy analysis) · Blacklow SC, Raines RT, Lim WA, Zamore PD, Knowles JR, Biochemistry 27:1158, 1988, PMID:3365378 (viscosity variation — diffusion-controlled) · Wierenga RK, Cell Mol Life Sci 67:3961–3982, 2010, PMID:20694739 (K_eq and diffusion-hit-limit values quoted in the drawer text; loop-6 lid) · BRENDA EC 5.3.1.1 S5 — UniProt P00942 (S. cerevisiae TPI1); residue numbers follow 1NEY/7TIM chain A PDB-author numbering (residues 2–248, shared by both structures) S6 — Repo display conventions: Rule 30 (narrated claims reconstructible from the scene) · Rule 34 geometry (7 geomClaims in tools/manifests/tpi.json) · every Å = a single crystallographic value (Precision vs. accuracy card) · [schematic] = TS-analog surrogate / same-skeleton re-annotation / stitched motion, a TIM transition state is never observed directly

› Backbones and ligand poses are taken directly from the listed PDB entries, superposed into one common frame (whole-Cα fit: 246 Cα, RMSD 0.285 Å). The Michaelis complex is the real 1NEY deposit; the enediolate is represented by the superposed 7TIM PGH transition-state analog; D-G3P is the same skeleton re-annotated in place (no G3P crystal is co-deposited here) — each is labeled as such in the narration. The inter-state transitions and the gold H⁺ path are schematic cartoons. Residue numbers follow the PDB-author numbering of 1NEY/7TIM chain A (S. cerevisiae, UniProt P00942).

N-halfC-halfONPMg/K