{"id":"20b172a0-4649-549b-b00d-702c0c6f2074","stable_key":"f8641d02-8413-5bd8-92e9-62ad49286e81:asa-ser516-is-the-site","predicate":"acetylates","statement":"When microsomal human cyclooxygenase-2 was incubated with acetyl-carbon-14 labelled aspirin the enzyme was acetylated, while an S516A mutant which retains enzyme activity was not acetylated, indicating that Ser-516 is the site of aspirin acetylation and is homologous to the active site serine of cyclooxygenase-1; an S516N mutant was catalytically active whereas an S516Q mutant lacked cyclooxygenase but retained peroxidase activity, and because in cyclooxygenase-1 the smaller asparagine substitution suffices to eliminate cyclooxygenase activity the active site of cyclooxygenase-2 is slightly larger.","claim_class":"mechanistic","status":"source_derived_draft","evidence_grade":"ungraded","direction":"positive","is_public":true,"mechanism_event_id":"f6214bd4-5862-53da-8d4a-cd7c6407d6ac","mechanism_event_label":"The labelled acetyl group lands on one serine, and the second enzyme’s pocket is roomier, which is why the two behave differently.","subject":{"id":"8ac405bf-3ea0-539a-8e5e-abf503dc7081","slug":"aspirin","display_name":"Aspirin / acetylsalicylic acid","entity_type_key":"drug"},"object":{"id":"48cd6cab-de3d-53bd-a690-a1f9e5b78a7d","slug":"cox2-ser516","display_name":"Serine 516 of cyclooxygenase-2","entity_type_key":"protein_state"},"evidence_count":1,"mechanism_event":{"id":"f6214bd4-5862-53da-8d4a-cd7c6407d6ac","stable_key":"f8641d02-8413-5bd8-92e9-62ad49286e81:asa-ser516-is-the-site-event","event_type":"biochemical_relationship","label":"The labelled acetyl group lands on one serine, and the second enzyme’s pocket is roomier, which is why the two behave differently.","description":"When microsomal human cyclooxygenase-2 was incubated with acetyl-carbon-14 labelled aspirin the enzyme was acetylated, while an S516A mutant which retains enzyme activity was not acetylated, indicating that Ser-516 is the site of aspirin acetylation and is homologous to the active site serine of cyclooxygenase-1; an S516N mutant was catalytically active whereas an S516Q mutant lacked cyclooxygenase but retained peroxidase activity, and because in cyclooxygenase-1 the smaller asparagine substitution suffices to eliminate cyclooxygenase activity the active site of cyclooxygenase-2 is slightly larger.","status":"provisional","compartment":null,"participants":[{"entity":{"id":"31ab0a39-42b1-52ce-a3e3-3ff05ad402bd","slug":"cox2-s516a","display_name":"Cyclooxygenase-2 with alanine substituted at position 516","entity_type_key":"protein_state"},"role":"unlabelled_mutant","stoichiometry":null,"state_label":"","sequence_order":0,"notes":""},{"entity":{"id":"d3e75f13-773e-5d31-a63a-c64e62371ed1","slug":"cox2-active-site-volume","display_name":"The volume of the cyclooxygenase-2 active site relative to that of cyclooxygenase-1","entity_type_key":"protein_state"},"role":"explaining_property","stoichiometry":null,"state_label":"","sequence_order":1,"notes":""},{"entity":{"id":"7a465c55-930d-54cf-89b3-952b2c16f558","slug":"cox1-ser530","display_name":"Serine 530 of cyclooxygenase-1","entity_type_key":"protein_state"},"role":"homologous_residue","stoichiometry":null,"state_label":"","sequence_order":2,"notes":""},{"entity":{"id":"8ac405bf-3ea0-539a-8e5e-abf503dc7081","slug":"aspirin","display_name":"Aspirin / acetylsalicylic acid","entity_type_key":"drug"},"role":"subject","stoichiometry":null,"state_label":"","sequence_order":3,"notes":""},{"entity":{"id":"48cd6cab-de3d-53bd-a690-a1f9e5b78a7d","slug":"cox2-ser516","display_name":"Serine 516 of cyclooxygenase-2","entity_type_key":"protein_state"},"role":"target","stoichiometry":null,"state_label":"","sequence_order":4,"notes":""}]},"contexts":[{"dimension":"evidence_span","value_text":"{\"source_cache\": \"artifacts/aspirin-research/8175750.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"9ba85af90c62b157cf53032375ca703e8b78eff6ddd22ad627bbd7e65965d8e9\", \"start_char\": 0, \"end_char\": 1786, \"text_sha256\": \"9ba85af90c62b157cf53032375ca703e8b78eff6ddd22ad627bbd7e65965d8e9\"}","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"experimental_model","value_text":"Human cyclooxygenase-1 and -2 expressed in cos-1 cells treated with aspirin and with carbon-14 labelled aspirin, with a mutant series at Ser-516","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"exposure","value_text":"Aspirin acetylation, with S516A, S516N, S516Q and S516M substitutions","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"limitations","value_text":"Identifies the site by direct labelling and explains the isoform difference by active site volume. Recombinant enzyme in a heterologous cell line.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"nutrient_topic","value_text":"Aspirin research collection; topical membership is not evidence of a direct clinical effect, and aspirin is recorded separately from salicylate, the metabolite it becomes.","comparator":null,"unit":null,"notes":"","entity":{"slug":"aspirin","display_name":"Aspirin / acetylsalicylic acid","entity_type_key":"drug"}},{"dimension":"organism","value_text":"Human enzyme","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"plain_language","value_text":"The labelled acetyl group lands on one serine, and the second enzyme’s pocket is roomier, which is why the two behave differently.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"primary_references","value_text":"[asa-p8175750] Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. (1994). https://pubmed.ncbi.nlm.nih.gov/8175750/ DOI: 10.1016/s0021-9258(17)36820-5","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"tissue_or_cell_type","value_text":"Recombinant cyclooxygenase","comparator":null,"unit":null,"notes":"","entity":null}],"evidence":[{"id":"ca38cc0d-7ee3-5562-b49c-3b76f7726c11","evidence_kind":"source_excerpt","locator":"Lines 221-232","start_line":221,"end_line":232,"excerpt":"### asa-ser516-is-the-site\nWhen microsomal human cyclooxygenase-2 was incubated with acetyl-carbon-14 labelled aspirin the enzyme was acetylated, while an S516A mutant which retains enzyme activity was not acetylated, indicating that Ser-516 is the site of aspirin acetylation and is homologous to the active site serine of cyclooxygenase-1; an S516N mutant was catalytically active whereas an S516Q mutant lacked cyclooxygenase but retained peroxidase activity, and because in cyclooxygenase-1 the smaller asparagine substitution suffices to eliminate cyclooxygenase activity the active site of cyclooxygenase-2 is slightly larger.\nCondition category: normal\nnutrient_topic: Aspirin research collection; topical membership is not evidence of a direct clinical effect, and aspirin is recorded separately from salicylate, the metabolite it becomes.\nplain_language: The labelled acetyl group lands on one serine, and the second enzyme’s pocket is roomier, which is why the two behave differently.\norganism: Human enzyme\ntissue_or_cell_type: Recombinant cyclooxygenase\nexperimental_model: Human cyclooxygenase-1 and -2 expressed in cos-1 cells treated with aspirin and with carbon-14 labelled aspirin, with a mutant series at Ser-516\nlimitations: Identifies the site by direct labelling and explains the isoform difference by active site volume. Recombinant enzyme in a heterologous cell line.\nexposure: Aspirin acetylation, with S516A, S516N, S516Q and S516M substitutions\nevidence_span: {\"source_cache\": \"artifacts/aspirin-research/8175750.abstract.txt\", \"locator\": \"Indexed abstract; zero-based, end-exclusive Unicode character offsets\", \"file_sha256\": \"9ba85af90c62b157cf53032375ca703e8b78eff6ddd22ad627bbd7e65965d8e9\", \"start_char\": 0, \"end_char\": 1786, \"text_sha256\": \"9ba85af90c62b157cf53032375ca703e8b78eff6ddd22ad627bbd7e65965d8e9\"}\n[asa-p8175750] Acetylation of human prostaglandin endoperoxide synthase-2 (cyclooxygenase-2) by aspirin. 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