{"id":"f74ca963-7f60-5b0f-87cb-797a6cd51a5b","stable_key":"7de430a5-1a36-5231-b931-35cc01d8f3bf:apap-potency-depends-on-peroxide","predicate":"inhibits","statement":"The potency of acetaminophen against both purified ovine cyclooxygenase-1 and human cyclooxygenase-2 was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione, giving half-maximal inhibitory concentrations of 33 and 980 micromolar respectively, acetaminophen was found to be a good reducing agent of both enzymes, and the results are consistent with a mechanism in which it reduces the active oxidized form of the enzyme to the resting form, so that inhibition would be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity.","claim_class":"mechanistic","status":"source_derived_draft","evidence_grade":"ungraded","direction":"negative","is_public":true,"mechanism_event_id":"8e6ec997-aba1-5a5c-b874-b89ac8c23d20","mechanism_event_label":"Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.","subject":{"id":"a901be21-7915-543c-ac67-c39ef72b0d03","slug":"acetaminophen","display_name":"Paracetamol","entity_type_key":"drug"},"object":{"id":"6284d26c-01e2-5e12-b192-ee391dd107e2","slug":"ptgs1","display_name":"Cyclooxygenase-1 (PTGS1)","entity_type_key":"protein"},"evidence_count":1,"mechanism_event":{"id":"8e6ec997-aba1-5a5c-b874-b89ac8c23d20","stable_key":"7de430a5-1a36-5231-b931-35cc01d8f3bf:apap-potency-depends-on-peroxide-event","event_type":"biochemical_relationship","label":"Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.","description":"The potency of acetaminophen against both purified ovine cyclooxygenase-1 and human cyclooxygenase-2 was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione, giving half-maximal inhibitory concentrations of 33 and 980 micromolar respectively, acetaminophen was found to be a good reducing agent of both enzymes, and the results are consistent with a mechanism in which it reduces the active oxidized form of the enzyme to the resting form, so that inhibition would be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity.","status":"provisional","compartment":null,"participants":[{"entity":{"id":"c8a31ed4-f2ca-5d43-8352-e9935129a785","slug":"ptgs2","display_name":"Cyclooxygenase-2 (PTGS2)","entity_type_key":"protein"},"role":"co_inhibited_isoform","stoichiometry":null,"state_label":"","sequence_order":0,"notes":""},{"entity":{"id":"a91446f5-a414-52d5-81b7-4e9a53d11614","slug":"glutathione-peroxidase-system","display_name":"Glutathione peroxidase with glutathione as a peroxide-removing system","entity_type_key":"cellular_process"},"role":"manipulation","stoichiometry":null,"state_label":"","sequence_order":1,"notes":""},{"entity":{"id":"b53aa1d7-8440-584c-aa8b-8a199aa832f1","slug":"peroxide-tone","display_name":"Ambient cellular hydroperoxide concentration","entity_type_key":"cellular_process"},"role":"governing_variable","stoichiometry":null,"state_label":"","sequence_order":2,"notes":""},{"entity":{"id":"db2a1421-bdbf-5d98-9292-0b308f88cdfc","slug":"reducing-cosubstrate","display_name":"A reducing cosubstrate of the peroxidase","entity_type_key":"cellular_process"},"role":"mechanism","stoichiometry":null,"state_label":"","sequence_order":3,"notes":""},{"entity":{"id":"ca772a9c-94e9-5072-a283-e25995682a15","slug":"pghs-peroxidase-site","display_name":"The peroxidase site of prostaglandin H synthase","entity_type_key":"protein_state"},"role":"site_of_action","stoichiometry":null,"state_label":"","sequence_order":4,"notes":""},{"entity":{"id":"a901be21-7915-543c-ac67-c39ef72b0d03","slug":"acetaminophen","display_name":"Paracetamol","entity_type_key":"drug"},"role":"subject","stoichiometry":null,"state_label":"","sequence_order":5,"notes":""},{"entity":{"id":"6284d26c-01e2-5e12-b192-ee391dd107e2","slug":"ptgs1","display_name":"Cyclooxygenase-1 (PTGS1)","entity_type_key":"protein"},"role":"target","stoichiometry":null,"state_label":"","sequence_order":6,"notes":""}]},"contexts":[{"dimension":"evidence_span","value_text":"{\"source_cache\": \"artifacts/paracetamol-research/11370851.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\", \"start_char\": 0, \"end_char\": 1086, \"text_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\"}","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"experimental_model","value_text":"Purified ovine cyclooxygenase-1 and human cyclooxygenase-2 assayed with and without a peroxide-removing system","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"exposure","value_text":"Acetaminophen with and without glutathione peroxidase and glutathione","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"limitations","value_text":"Adding a peroxide-removing system is the manipulation that reveals the mechanism, because it changes potency thirtyfold without changing the drug. Purified enzyme, so the concentrations are not tissue concentrations.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"nutrient_topic","value_text":"Paracetamol research collection; topical membership is not evidence of a direct clinical effect, and the drug is recorded separately from the metabolites NAPQI and AM404.","comparator":null,"unit":null,"notes":"","entity":{"slug":"acetaminophen","display_name":"Paracetamol","entity_type_key":"drug"}},{"dimension":"organism","value_text":"Sheep and human enzyme","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"plain_language","value_text":"Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"primary_references","value_text":"[apap-p11370851] Mechanism of acetaminophen inhibition of cyclooxygenase isoforms. (2001). https://pubmed.ncbi.nlm.nih.gov/11370851/ DOI: 10.1006/abbi.2000.2232","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"tissue_or_cell_type","value_text":"Purified cyclooxygenase","comparator":null,"unit":null,"notes":"","entity":null}],"evidence":[{"id":"0e6eae65-6e8c-5dea-8802-c6fb87510360","evidence_kind":"source_excerpt","locator":"Lines 77-88","start_line":77,"end_line":88,"excerpt":"### apap-potency-depends-on-peroxide\nThe potency of acetaminophen against both purified ovine cyclooxygenase-1 and human cyclooxygenase-2 was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione, giving half-maximal inhibitory concentrations of 33 and 980 micromolar respectively, acetaminophen was found to be a good reducing agent of both enzymes, and the results are consistent with a mechanism in which it reduces the active oxidized form of the enzyme to the resting form, so that inhibition would be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity.\nCondition category: normal\nnutrient_topic: Paracetamol research collection; topical membership is not evidence of a direct clinical effect, and the drug is recorded separately from the metabolites NAPQI and AM404.\nplain_language: Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.\norganism: Sheep and human enzyme\ntissue_or_cell_type: Purified cyclooxygenase\nexperimental_model: Purified ovine cyclooxygenase-1 and human cyclooxygenase-2 assayed with and without a peroxide-removing system\nlimitations: Adding a peroxide-removing system is the manipulation that reveals the mechanism, because it changes potency thirtyfold without changing the drug. Purified enzyme, so the concentrations are not tissue concentrations.\nexposure: Acetaminophen with and without glutathione peroxidase and glutathione\nevidence_span: {\"source_cache\": \"artifacts/paracetamol-research/11370851.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\", \"start_char\": 0, \"end_char\": 1086, \"text_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\"}\n[apap-p11370851] Mechanism of acetaminophen inhibition of cyclooxygenase isoforms. (2001). https://pubmed.ncbi.nlm.nih.gov/11370851/ DOI: 10.1006/abbi.2000.2232","model_system":"Purified ovine cyclooxygenase-1 and human cyclooxygenase-2 assayed with and without a peroxide-removing system","directness":"author_interpretation","verification_status":"source_derived_draft","notes":"Exact curation-document quotation, not publisher quotation. Study references: [apap-p11370851] Mechanism of acetaminophen inhibition of cyclooxygenase isoforms. (2001). https://pubmed.ncbi.nlm.nih.gov/11370851/ DOI: 10.1006/abbi.2000.2232","relationship":"supports","weight":1.0,"link_notes":"","source":{"id":"9ee11177-f860-5877-bb82-f4ee4acdc538","stable_key":"import-7de430a5-1a36-5231-b931-35cc01d8f3bf","title":"Paracetamol: the enzyme it reduces rather than blocks, the isoform that turned out not to exist, the metabolite that carries the analgesia, and the metabolite that destroys the liver (2026-09-22)","document_type":"imported_text","citation_label":"AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text.","file_path":"","sha256":"9c145887aa10fa7b85f54712e0b41fd9e04fb9544d3182793dd84414dbf8c2ae","revision_id":"c3e7827a-4f10-55e0-97b9-5782b044f7cc","review_status":"unverified_draft","notes":""}}],"relations":[],"conflicts":[{"id":"e665f666-5b75-5292-9bff-37a7ec2bfb07","title":"Does paracetamol act at the peroxidase site or the cyclooxygenase site?","kind":"contradiction","status":"open","why":"The peroxidase account is strongly supported: paracetamol is a good reducing agent of both isoforms, removing peroxide with glutathione peroxidase makes it thirtyfold more potent, and adding hydroperoxide back abolishes inhibition in cells, with 0.3 micromolar 12-hydroperoxyeicosatetraenoic acid enough to reverse it completely. Against that, phenolic compounds including paracetamol still inhibit prostaglandin H synthase reconstituted with manganese protoporphyrin, which retains cyclooxygenase but almost no peroxidase activity, their inhibition depends strongly on arachidonic acid concentration, and adding 10 micromolar prostaglandin G2 did not prevent it; those authors concluded the compounds compete with arachidonate at the cyclooxygenase site in addition to or independent of any effect on peroxide tone. A third record shows the two sites are connected, since cyclooxygenase activity depends on oxidation of an active-site tyrosine by electron transfer from the peroxidase, so a drug that reduces one site can quench the radical at the other. The accounts are not exclusive and no record here measures their relative contribution at a therapeutic concentration.","resolution":"Unresolved; needs review.","created_at":"2026-09-22 17:05:56","record_type":"conflict","display_label":"Recorded conflict","record_url":"/conflicts/e665f666-5b75-5292-9bff-37a7ec2bfb07","sides":[{"conflict_id":"e665f666-5b75-5292-9bff-37a7ec2bfb07","ordinal":0,"label":"Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.","revision_id":"c3e7827a-4f10-55e0-97b9-5782b044f7cc","start_line":77,"end_line":88,"quote":"### apap-potency-depends-on-peroxide\nThe potency of acetaminophen against both purified ovine cyclooxygenase-1 and human cyclooxygenase-2 was increased approximately 30-fold by the presence of glutathione peroxidase and glutathione, giving half-maximal inhibitory concentrations of 33 and 980 micromolar respectively, acetaminophen was found to be a good reducing agent of both enzymes, and the results are consistent with a mechanism in which it reduces the active oxidized form of the enzyme to the resting form, so that inhibition would be more effective under conditions of low peroxide concentration, consistent with the known tissue selectivity.\nCondition category: normal\nnutrient_topic: Paracetamol research collection; topical membership is not evidence of a direct clinical effect, and the drug is recorded separately from the metabolites NAPQI and AM404.\nplain_language: Take the peroxide away and the same drug becomes thirty times stronger, which is the whole mechanism in one experiment.\norganism: Sheep and human enzyme\ntissue_or_cell_type: Purified cyclooxygenase\nexperimental_model: Purified ovine cyclooxygenase-1 and human cyclooxygenase-2 assayed with and without a peroxide-removing system\nlimitations: Adding a peroxide-removing system is the manipulation that reveals the mechanism, because it changes potency thirtyfold without changing the drug. Purified enzyme, so the concentrations are not tissue concentrations.\nexposure: Acetaminophen with and without glutathione peroxidase and glutathione\nevidence_span: {\"source_cache\": \"artifacts/paracetamol-research/11370851.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\", \"start_char\": 0, \"end_char\": 1086, \"text_sha256\": \"4eeda4b163828f2cc2a4341c4810df6bf6c683d6165eeb09479104f185e52ca4\"}\n[apap-p11370851] Mechanism of acetaminophen inhibition of cyclooxygenase isoforms. (2001). https://pubmed.ncbi.nlm.nih.gov/11370851/ DOI: 10.1006/abbi.2000.2232","source_key":"import-7de430a5-1a36-5231-b931-35cc01d8f3bf","source_title":"Paracetamol: the enzyme it reduces rather than blocks, the isoform that turned out not to exist, the metabolite that carries the analgesia, and the metabolite that destroys the liver (2026-09-22)","claim_ids":["f74ca963-7f60-5b0f-87cb-797a6cd51a5b"]},{"conflict_id":"e665f666-5b75-5292-9bff-37a7ec2bfb07","ordinal":1,"label":"In an enzyme stripped of its peroxidase the drug still works, which argues it also competes at the other site.","revision_id":"c3e7827a-4f10-55e0-97b9-5782b044f7cc","start_line":155,"end_line":166,"quote":"### apap-maybe-the-cox-site-instead\nHigher concentrations of eugenol were required to inhibit iron-reconstituted prostaglandin H synthase than the manganese-reconstituted enzyme which retains cyclooxygenase but not peroxidase activity, inhibition was highly dependent on arachidonic acid concentration, adding 10 micromolar prostaglandin G2 did not prevent the inhibitory effects, and other phenolic compounds including guaiacol, butylated hydroxyanisole and acetaminophen inhibited the manganese enzyme similarly, demonstrating that these compounds specifically inhibit the cyclooxygenase component in addition to or independent of their effect on peroxide tone, which the authors suggest is due to competition with arachidonic acid for the active site.\nCondition category: normal\nnutrient_topic: Paracetamol research collection; topical membership is not evidence of a direct clinical effect, and the drug is recorded separately from the metabolites NAPQI and AM404.\nplain_language: In an enzyme stripped of its peroxidase the drug still works, which argues it also competes at the other site.\norganism: Enzyme\ntissue_or_cell_type: Reconstituted prostaglandin H synthase\nexperimental_model: Prostaglandin H synthase apoenzyme reconstituted with manganese or iron protoporphyrin\nlimitations: The manganese-reconstituted enzyme retains cyclooxygenase but little peroxidase activity, which is what lets this study separate the two proposed sites. Its conclusion is the opposite of the peroxide-tone account.\nexposure: Eugenol, guaiacol, butylated hydroxyanisole and acetaminophen against both reconstituted forms, with prostaglandin G2 added\nevidence_span: {\"source_cache\": \"artifacts/paracetamol-research/2511429.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"8dbe8c6f23fc857be6bba07e2a518c59f8dc76c9f8625c25ba84030c43f6e636\", \"start_char\": 0, \"end_char\": 1593, \"text_sha256\": \"8dbe8c6f23fc857be6bba07e2a518c59f8dc76c9f8625c25ba84030c43f6e636\"}\n[apap-p2511429] Mechanism of inhibition of prostaglandin H synthase by eugenol and other phenolic peroxidase substrates. (1989). https://pubmed.ncbi.nlm.nih.gov/2511429/ DOI: 10.1016/s0026-895x(25)09658-0","source_key":"import-7de430a5-1a36-5231-b931-35cc01d8f3bf","source_title":"Paracetamol: the enzyme it reduces rather than blocks, the isoform that turned out not to exist, the metabolite that carries the analgesia, and the metabolite that destroys the liver (2026-09-22)","claim_ids":["e283ddbf-0b21-5174-a738-f19e14ab5174"]}]}],"corrections":[],"research":null}