{"id":"dfe5cab1-3955-564f-b9c1-29dfaf732a76","stable_key":"33bef151-bdc7-5892-aff6-0345ab149739:removing-those-bonds-does-not-change-substrate-binding","predicate":"does_not_change","statement":"The same four substitutions left Km essentially unchanged, so the bonds support catalysis rather than ground-state substrate binding.","claim_class":"mechanistic","status":"source_derived_draft","evidence_grade":"ungraded","direction":"neutral","is_public":true,"mechanism_event_id":"29d86828-179f-5756-bcba-76fb3eb1e63c","mechanism_event_label":"The same four substitutions left Km essentially unchanged, so the bonds support catalysis rather than ground-state substrate binding.","subject":{"id":"43ae455c-80d0-5beb-be77-15001c6d3d57","slug":"nattokinase-hydrogen-bond-mutants","display_name":"Nattokinase Ser33Ala, Asp60Ala, Ser62Ala and Thr220Ala mutants","entity_type_key":"protein_state"},"object":{"id":"041ab093-2517-5839-a8a2-95e11a631123","slug":"nattokinase-substrate-binding","display_name":"Nattokinase ground-state substrate binding","entity_type_key":"cellular_process"},"evidence_count":1,"mechanism_event":{"id":"29d86828-179f-5756-bcba-76fb3eb1e63c","stable_key":"33bef151-bdc7-5892-aff6-0345ab149739:removing-those-bonds-does-not-change-substrate-binding-event","event_type":"biochemical_relationship","label":"The same four substitutions left Km essentially unchanged, so the bonds support catalysis rather than ground-state substrate binding.","description":"Enzyme kinetics with molecular dynamics and free-energy perturbation","status":"provisional","compartment":null,"participants":[{"entity":{"id":"43ae455c-80d0-5beb-be77-15001c6d3d57","slug":"nattokinase-hydrogen-bond-mutants","display_name":"Nattokinase Ser33Ala, Asp60Ala, Ser62Ala and Thr220Ala mutants","entity_type_key":"protein_state"},"role":"acting component","stoichiometry":null,"state_label":"","sequence_order":0,"notes":""},{"entity":{"id":"041ab093-2517-5839-a8a2-95e11a631123","slug":"nattokinase-substrate-binding","display_name":"Nattokinase ground-state substrate binding","entity_type_key":"cellular_process"},"role":"component the finding is about","stoichiometry":null,"state_label":"","sequence_order":1,"notes":""}]},"contexts":[{"dimension":"availability_state","value_text":"machinery_impairment","comparator":null,"unit":null,"notes":"Imported condition classification; unverified.","entity":null},{"dimension":"duration","value_text":"Not stated here","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"evidence_access","value_text":"Primary PubMed abstract and indexed metadata reviewed. 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Simulation partially released Asp32, His64 and Asn155 in the mutants, which is the proposed explanation rather than a separate measurement.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"organism","value_text":"Recombinant subtilisin NK mutants","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"plain_language","value_text":"The same four substitutions left Km essentially unchanged, so the bonds support catalysis rather than ground-state substrate binding.","comparator":null,"unit":null,"notes":"","entity":null},{"dimension":"primary_references","value_text":"Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation. 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Every reference in that document was resolved against live PubMed with its abstract read and its DOI cross-checked on 2026-09-23, and the EFSA novel-food opinion was retrieved and read in full. That check corrected two PMIDs that pointed at unrelated papers, two DOIs, and two papers recorded as carrying no erratum that do carry one; it also reversed three findings the supplied document had stated backwards. Two papers carry a published correction, recorded as such and not as a retraction. Three sources are not indexed in PubMed and are cited by what they have. Laboratory lineages are recorded, so the four papers from one group, the three from another and the two readings of a single applicant dossier cannot be counted as separate lines of support. Study-specific doses, units, populations and limitations retained; activity units are never converted between systems. 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