Liver Glycogen Phosphorylase - 3D Mechanism Simulator

START HERE — how to read glycogen(n) + Pi → glycogen(n−1) + G1P A single phosphorolysis cartoon on the human liver GP pocket. Real crystal: 1FA9 (2.4 Å), form a, chain A, with covalent PLP on PDB Lys680 (UniProt 681), a catalytic-site GLC inhibitor, and allosteric AMP. Crystal GLC is not glycogen and not G1P — it is a named analog of the non-reducing terminal glucose [schematic]. Pi is not deposited on 1FA9; 3CEH PO4 is the wrong site (~30 Å from C1) and is not used. The phosphate of PLP is commonly drawn as the catalytic acid/base — but that assignment is disputed in the primary literature, so read “Is the PLP phosphate the proton donor?” before treating ③–⑤ as fact. Lys680 tethers PLP and is not the sugar nucleophile. Gold H⁺ parks on analog O1 (page staging — the proton donor and its timing are both unsettled); white is the phosphoryl (PO₃) transfer, not a hydride. Schematic Pi/G1P distances are not geomClaims. AMP ≠ catalytic pocket. PYGB / PYGM are other genes.
Crystal structures PDB 1FA9 — human liver glycogen phosphorylase a, 2.4 Å, PLP + GLC + AMP (Rath 2000). Coordinates are public. There is no human catalytic Pi on this frame. 8EMS is PLP-only EM. 3CEH / 1L5Q / 2ATI are drug complexes (NBG analog, not glycogen). 5IKO is PYGB. 1Z8D is PYGM without PLP. Rabbit-muscle GP crystals are mechanism literature, not this mesh.
Mechanism — what is settled, and what is still disputed in the literature Structural source: Rath et al., Chem Biol 2000, PMID:10949035 — human liver GP a with glucose and AMP. This paper supplies the coordinates on this page; it is a structure paper and does not settle the chemical step. Settled (all models agree): the substrate is inorganic Pi, not water (phosphorolysis, EC 2.4.1.1); the product is α-D-glucose 1-phosphate; PLP is covalently tethered to Lys680 and does not leave; there is no covalent glycosyl-enzyme intermediate; the glycosidic oxygen must carry a proton in the net reaction. Not settled: which group actually delivers that proton. Two incompatible models have stood since 1982 and neither has been eliminated — see the next entry, “Is the PLP phosphate the proton donor?” Source: Rath 2000 (structure) · UniProt P06737 · Rhea 41732 · EC 2.4.1.1
Is the PLP phosphate the proton donor? — an open question, stated in full This is the one mechanistic point on this page where the primary literature disagrees with itself. Nothing in the three models below is a settled result, and this page does not present any of them as settled. Constraint both models accept. Catalytically competent GP requires PLP in its dianionic form and requires Pi (or arsenate) to be present — shown with the D-glucal substrate, which is consumed only when an anion is bound. Klein et al. rationalize this as “a proton transfer relay involving juxtaposed phosphates”. Klein, Palm & Helmreich, Biochemistry 1982;21(26):6675–6684 · PMID:7159553 · doi:10.1021/bi00269a010 Model A — Pi-mediated proton relay (general acid/base). The PLP 5′-phosphate acts as a general acid/base, and the proton reaches the glycosidic oxygen through the substrate Pi, not directly from PLP. Klein, Palm & Helmreich 1982 (above) · Klein, Im, Palm & Helmreich, “Does pyridoxal 5′-phosphate function in glycogen phosphorylase as an electrophilic or a general acid catalyst?”, Biochemistry 1984;23(24):5455–5462 · PMID:6098301 · Palm, Klein, Schächtele & Helmreich, “The role of pyridoxal 5′-phosphate in glycogen phosphorylase catalysis”, Biochemistry 1990;29(5):1099–1107 · doi:10.1021/bi00457a001 Model B — electrophilic catalyst, no discrete glycosidic protonation. The PLP phosphate acts as an electrophilic catalyst that stabilizes the transition state; the reaction shows no pKa dependence, and no proton is delivered to the glycosidic oxygen as a separate step. Withers, Shechosky & Madsen, “Pyridoxal phosphate is not the acid catalyst in the glycogen phosphorylase catalytic mechanism”, Biochem Biophys Res Commun 1982;108(1):322–328 · doi:10.1016/0006-291x(82)91869-1 · Takagi, Fukui & Shimomura, “Catalytic mechanism of glycogen phosphorylase: pyridoxal(5′)diphospho(1)-α-D-glucose as a transition-state analogue”, PNAS 1982;79(12):3716–3719 · doi:10.1073/pnas.79.12.3716 · Withers, Madsen, Sykes, Takagi, Shimomura & Fukui, “Evidence for direct phosphate-phosphate interaction between pyridoxal phosphate and substrate in the glycogen phosphorylase catalytic mechanism”, JBC 1981;256(21):10759–10762 · doi:10.1016/S0021-9258(19)68505-4 Modern computation — routes the proton through Pi and returns it to PLP. QM/MM gives: PLP phosphate → Pi; Pi protonates the glycosidic oxygen; Pi attacks C1 with the proton returning to PLP concomitantly. There is no parked, long-lived protonated glycosidic intermediate. Guariguata et al., ChemMedChem 2018 · PMID:29905983 · doi:10.1002/cmdc.201800218 What this page actually draws is a fourth thing, and it is none of the three above. ③ shows a direct PLP O2P → glycosidic O1 transfer with the proton parking on the oxygen, and ④ then shows Pi attacking C1 as a separate later event. That is not Model A (which relays through Pi, not directly from PLP), not Model B (which has no discrete glycosidic-oxygen protonation at all), and not the QM/MM result (which returns the proton to PLP rather than parking it on O1). It is a deliberate pedagogical staging, chosen so that one arrow equals one teachable event. It carries the (hypothetical, lit.-based) tag at the step for exactly this reason, and it should be read as a diagram of the open question, not as an answer to it. On timing: there is no measured number, for any of the four. No sub-step timing that separates protonation from C1 attack has been measured in any of these studies. A steady-state kcat is a turnover rate for the entire cycle and is not a duration for an individual chemical step. 1FA9 cannot settle it either — at 2.4 Å no catalytic proton is deposited at that position. The on-screen gap between ③ and ④ is therefore a staging interval with no timescale attached, and this page makes no claim that the two events are separable in real time.
0 — Setup: PLP + AMP stay, catalytic pocket shown empty 1FA9 holds PLP (Lys680), catalytic GLC, and allosteric AMP. The catalytic pocket is shown empty so ordered binding can start. PLP stays visible as a prosthetic occupant. AMP stays at the allosteric site. Opaque, no autoplay. Crystal GLC is an inhibitor analog, not glycogen.Source: PDB 1FA9
① — Terminal glucose presented (GLC analog) Crystal GLC docks as a named analog of the glycogen non-reducing end — [schematic], not a polymer. C1 faces the Pi seat. His377 ND1 ⋯ GLC O2 = 2.86 Å on this deposit. Crystal O1 is the inhibitor OH, not the glycogen glycosidic oxygen.Source: 1FA9 GLC pose
② — Inorganic phosphate binds Pi is seated between GLC C1 and PLP-PO₄ — [schematic], not a deposited ternary. 3CEH PO4 is ~30 Å from NBG C1 and is rejected. Product of this enzyme is G1P, not G6P (that is PGM1).Source: no human catalytic Pi on 1FA9; 3CEH PO4 discarded
③ — [inferred, disputed] PLP phosphate → glycosidic O1, drawn as a discrete parked proton (hypothetical, lit.-based — the proton donor is disputed) A gold H⁺ sphere rides a gold arrow from PLP O2P to analog O1 and parks there. Lys680 NZ ⋯ PLP C4A = 1.45 Å is a measured crystal fact, not an inference. What is disputed: whether the PLP phosphate donates this proton at all, and whether the proton arrives directly from PLP or via the substrate Pi. Klein/Palm 1982 & 1984 place it in a relay through juxtaposed phosphates; Withers 1982 and Takagi 1982 argue PLP is an electrophilic catalyst with no discrete glycosidic-oxygen protonation; QM/MM (Guariguata 2018) routes the proton through Pi and returns it to PLP, so it never parks on O1. This step is a teaching staging of that open dispute, not a resolution of it.Sources: measured 1FA9 LINK (Lys680 ⋯ PLP C4A = 1.45 Å) · Klein 1982 PMID:7159553 · Klein 1984 PMID:6098301 · Withers 1982 doi:10.1016/0006-291x(82)91869-1 · Takagi 1982 doi:10.1073/pnas.79.12.3716 · Guariguata 2018 PMID:29905983 — full discussion under “Is the PLP phosphate the proton donor?”
④ — Pi attacks C1 → G1P (hypothetical, lit.-based) White phosphoryl rides from Pi O to analog C1. GLC + Pi fade in place to schematic G1P. Parked gold H⁺ stays on analog O1. Schematic Pi⋯C1 is not a geomClaim. Slow-motion on purpose. No covalent glycosyl-enzyme.Sources: settled — the substrate is inorganic Pi, not water (phosphorolysis, EC 2.4.1.1), and the product is α-D-glucose 1-phosphate (UniProt P06737 · Rhea 41732); all cited models agree Pi attacks C1 and no covalent glycosyl-enzyme forms. Staging only — that this attack is drawn after a separate, parked protonation (③) is a page choice for one-arrow-one-event teaching; Takagi 1982 treats protonation and attack as a single concerted event, and QM/MM (Guariguata 2018) treats them as concomitant. See “Is the PLP phosphate the proton donor?”
⑤ — PLP regenerates (hypothetical, lit.-based) The parked gold H⁺ leaves. PLP does not leave. Net phosphorolysis still needs that proton on the glycosidic oxygen. Explicitly: this departure is drawn only because ③ parked the proton there; it is a bookkeeping consequence of this page's staging, not an independently observed or separately timed step, and no cited study resolves where the proton physically goes at this point.Sources: EC 2.4.1.1 net accounting (stoichiometry settled) · the drawn departure order is page staging, not literature · QM/MM (Guariguata 2018) instead returns the proton to PLP concomitantly with C1 attack
⑥ — G1P leaves — PGM1 is next [schematic] G1P departs. PLP stays on Lys680. AMP stays allosteric. G1P is the substrate of PGM1; G6P is not made here.Source: glycogen-breakdown bookkeeping

How to read this page — a first-pass primer First pass: Setup (PLP + AMP present, catalytic pocket empty, Lys680 ★) → ① GLC analog docks → ② schematic Pi docks → ③ gold H⁺ O2P → O1 (parks) → ④ white phosphoryl Pi-O → C1, GLC+Pi become G1P → ⑤ gold H⁺ leaves → ⑥ G1P leaves; PLP and AMP stay. Second pass: 2.86 Å and 1.45 Å are crystal facts on 1FA9; Pi, G1P, and the H spheres are [schematic]. Third pass: open “Disagreements” for analog vs glycogen, 3CEH PO4, numbering −1, and concerted vs split. Fourth pass — required before trusting ③–⑤: open “Is the PLP phosphate the proton donor?” That entry separates what is settled (Pi is the substrate, G1P is the product, no covalent glycosyl-enzyme) from what is disputed (which group delivers the glycosidic proton), names the two competing 1982 models and the 2018 QM/MM result with citations, and states plainly that this page’s drawing is a fourth, pedagogical staging that matches none of the three exactly — and that no sub-step timing has ever been measured for any of them.
Chemistry primer — phosphorolysis, not hydrolysis Glycogen phosphorylase uses Pi, not water, to clip the non-reducing α-1,4 end. The anomeric carbon (C1) is attacked by a phosphate oxygen; the glycosidic oxygen carries a proton in the net reaction. Who delivers that proton is the disputed point — the PLP 5′-phosphate, the substrate Pi as a relay, or no discrete delivery at all if PLP acts as an electrophilic catalyst. This page draws the first option as a teaching staging; see “Is the PLP phosphate the proton donor?” for the three-way disagreement and its citations. Product is α-D-glucose 1-phosphate, which PGM1 later isomerizes to G6P. Gold = H⁺. White = phosphoryl transfer (not hydride). This is not a hydrolase.
Words used on screen — analog · prosthetic PLP · allosteric AMP Analog — 1FA9 GLC is a competitive inhibitor at the catalytic site (T-state stabilizer), used here as a stand-in for the terminal glucose. Prosthetic PLP — covalently on Lys680; stays. Allosteric AMP — activator at PDB 42–44 / 75 / 309; named, not this chemistry. PDB numbering = UniProt P06737 minus 1 (Lys681 → 680, Ser15 → SEP 14).

Legend & Fidelity

Mechanism fidelity — measured vs. inferred The measured layer: 1FA9 (2.4 Å) with PLP, GLC, AMP; His377 ND1 ⋯ GLC O2 = 2.86 Å; Lys680 NZ ⋯ PLP C4A = 1.45 Å; PLP O2P ⋯ GLC C1 = 4.21 Å; Arg309 NH2 ⋯ AMP O2P = 2.70 Å. Every number in that sentence is a distance read off deposited coordinates. The inferred layer ([schematic]): Pi body, G1P, transferring H⁺/nucleophile spheres, product leave, and ⑤ PLP regeneration. The inferred layer is graded, and the grade differs by step: the substrate identity and product identity are settled (Pi in, α-D-glucose 1-phosphate out, no covalent glycosyl-enzyme), whereas the ③ proton transfer and its position in the sequence are actively disputed in the primary literature and are not merely unsourced — they are contested, with citations under “Is the PLP phosphate the proton donor?”. Binding order is staging, not a kinetic fit. The protein mesh is static. Schematic Pi contacts are not geomClaims.
Disagreements on record (a) GLC ≠ glycogen ≠ G1P. Crystal glucose is an inhibitor analog. (b) No human catalytic Pi. 3CEH PO4 is ~30 Å from sugar C1 — wrong site, not overlaid. (c) Concerted vs split. Takagi, Fukui & Shimomura (PNAS 1982;79:3716–3719, doi) present protonation and Pi attack as a single concerted event via a pyridoxal-diphospho-glucose transition-state analogue; QM/MM (Guariguata 2018, PMID:29905983) also treats them as concomitant. This page splits them (③ then ④) purely so one arrow equals one teachable event. (d) Numbering. PDB = UniProt − 1; labels are PDB ids. (e) Species. The mechanism literature is overwhelmingly rabbit muscle GP; this mesh is human liver. The dispute over the proton donor was settled — as a dispute — on rabbit muscle enzyme, and no human-liver-specific determination of the proton donor is cited here. (f) PYGB / PYGM are other genes. (g) Form a phosphorylation (SEP 14 / UniProt Ser15) is regulation, not motion. (h) Who protonates the glycosidic oxygen is unresolved. Klein/Palm/Helmreich 1982 & 1984 (relay through Pi) vs Withers/Shechosky/Madsen 1982 and Takagi 1982 (electrophilic catalyst, PLP is not the acid) vs Guariguata 2018 (through Pi, proton returns to PLP). Both camps have experimental support; neither is refuted. The page’s ③–⑤ depicts a fourth reading (direct PLP→O1 with a parked proton) that matches none of the three exactly, and is labeled as staging. (i) No sub-step timing exists. No cited study measures the interval between protonation and C1 attack. A steady-state kcat is a whole-cycle turnover rate, not a chemical-step duration; at 2.4 Å, 1FA9 deposits no catalytic proton. The ③→④ gap on screen carries no timescale and does not assert the events are separable in time. See “Is the PLP phosphate the proton donor?” for full citations.
Regulation — AMP · phospho-Ser · isoforms AMP is an allosteric activator at a site distinct from the catalytic pocket (Arg309 NH2 ⋯ AMP O2P = 2.70 Å) — named, not animated as catalytic motion. Form a = phospho-Ser (UniProt 15 = PDB SEP 14). Cytosolic liver PYGL (this page) vs muscle PYGM vs brain PYGB are different genes. GSD VI (Hers) is named, not simulated.Source: UniProt P06737 BINDING · Rath 2000 PMID:10949035
Kinetics scope (no measured timecourse on this page) One glycogen(n) + Pi → glycogen(n−1) + G1P event is drawn, not a steady-state run. Compiled BRENDA values for EC 2.4.1.1 are not re-asserted here — no measured k_cat/K_m on this page [schematic]. Pair with a kinetics chapter.Source: BRENDA EC 2.4.1.1 — no k_cat/K_m re-asserted
Sequence Human PYGL UniProt P06737 · NCBI Gene 5836. Screen uses 1FA9 PDB-author numbering (Lys680 = UniProt 681). Chain A of a dimer biological unit.
Simulation map — what you see is what is written Setup = 1FA9 catalytic pocket empty, PLP + AMP visible (opaque, no autoplay). ① GLC analog moves in (crystal). ② Pi moves in (schematic). ③ gold H⁺ + gold arrow PLP O2P → analog O1 (parks). ④ slow-mo: white phosphoryl + white arrow Pi-O → C1; GLC+Pi fade to G1P; parked gold H⁺ remains. ⑤ gold H⁺ leaves. ⑥ G1P leaves; PLP and AMP stay. Only these events move.
Sources — bibliography of this page S1 — PDB 1FA9: human liver GP a, 2.4 Å, PLP + GLC + AMP — public coordinates; measured NZ⋯C4A = 1.45 Å · ND1⋯O2 = 2.86 Å S2 — Rath et al., Chem Biol 2000, PMID:10949035 — human liver GP a with glucose and AMP S3 — Sequence: UniProt P06737 (human PYGL) · NCBI Gene 5836 · Rhea 41732 S4 — Kinetics: BRENDA EC 2.4.1.1 — no k_cat/K_m re-asserted on this page

› 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., catalytic Pi on 1FA9, G1P identity, H⁺ flight, product release), the pose is drawn schematically and labeled as such in the narration.