Steric blockade of a substrate channel by an introduced side chain

evidence_span

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experimental_model

Computational modelling of oxygenation stereospecificity in native and aspirin-acetylated cyclooxygenase-2

exposure

A mechanistic hypothesis tested against experimental oxygenation patterns

limitations

A computational model reproducing known patterns, not a new measurement. It offers an explanation for the switch rather than evidence that it occurs.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Enzyme

plain_language

The same bulk that blocks the first enzyme explains why the second one makes mirror-image products.

primary_references

[asa-p23786234] A mechanistic hypothesis for the aspirin-induced switch in lipid mediator production by cyclooxygenase-2. (2013). https://pubmed.ncbi.nlm.nih.gov/23786234/ DOI: 10.1021/ja402870k

tissue_or_cell_type

Cyclooxygenase-2

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 351–362

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Computational modelling of oxygenation stereospecificity in native and aspirin-acetylated cyclooxygenase-2 · source_derived_draft · unverified_draft

### asa-steric-shielding-explains-stereochemistry A mechanistic hypothesis identifying steric shielding as the main determinant of oxygenation stereospecificity in cyclooxygenase-2 is supported by a computational model which accurately reproduces experimental oxygenation patterns on both native and aspirin-inhibited enzyme, addressing why Ser530 acetylation shifts the stereochemistry and relative abundance of generated products. Condition category: normal nutrient_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. plain_language: The same bulk that blocks the first enzyme explains why the second one makes mirror-image products. organism: Enzyme tissue_or_cell_type: Cyclooxygenase-2 experimental_model: Computational modelling of oxygenation stereospecificity in native and aspirin-acetylated cyclooxygenase-2 limitations: A computational model reproducing known patterns, not a new measurement. It offers an explanation for the switch rather than evidence that it occurs. exposure: A mechanistic hypothesis tested against experimental oxygenation patterns evidence_span: {"source_cache": "artifacts/aspirin-research/23786234.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "bdf3d33c4cc14fc4b2190180d78b68971144966047e654abb59b9ca7a83ab824", "start_char": 0, "end_char": 834, "text_sha256": "bdf3d33c4cc14fc4b2190180d78b68971144966047e654abb59b9ca7a83ab824"} [asa-p23786234] A mechanistic hypothesis for the aspirin-induced switch in lipid mediator production by cyclooxygenase-2. (2013). https://pubmed.ncbi.nlm.nih.gov/23786234/ DOI: 10.1021/ja402870k

evidence_span

{"source_cache": "artifacts/aspirin-research/26859324.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "2d5dab1d6b7576a909c9cef6b5c247e6a399946afbf7ef6d428d67475a363c34", "start_char": 0, "end_char": 1553, "text_sha256": "2d5dab1d6b7576a909c9cef6b5c247e6a399946afbf7ef6d428d67475a363c34"}

experimental_model

Crystal structures of an S530T murine COX-2 mutant, aspirin-acetylated human COX-2 and human COX-2 with salicylate, at 1.9 to 2.4 angstrom

exposure

Aspirin acetylation and salicylate binding, with an S530T/G533V double mutant and purification in several detergent systems

limitations

Much higher resolution than the 1995 structure and it captures salicylate separately. The authors note that detergent and lipid in the membrane-binding domain change the acetylation rate in vitro, which is a caution about all such measurements.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Mouse and human enzyme

plain_language

At high resolution the acetyl group seals the groove completely, and salicylate is caught sitting where aspirin sits before it reacts.

primary_references

[asa-p26859324] Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. (2016). https://pubmed.ncbi.nlm.nih.gov/26859324/ DOI: 10.1021/acs.biochem.5b01378

tissue_or_cell_type

Crystallised cyclooxygenase-2

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 156–167

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Crystal structures of an S530T murine COX-2 mutant, aspirin-acetylated human COX-2 and human COX-2 with salicylate, at 1.9 to 2.4 angstrom · source_derived_draft · unverified_draft

### asa-acetyl-blocks-the-groove The crystal structures reveal that the acetylated Ser-530 completely blocks access to the hydrophobic groove, that the observed binding pose of salicylate is reflective of the enzyme-inhibitor complex prior to acetylation, and that the observed Thr-530 rotamer in the S530T mutant does not impede access to the groove; differential acetylation of COX-2 purified in various detergent systems and nanodiscs indicates that detergent and lipid binding within the membrane-binding domain alters the rate of the acetylation reaction in vitro. Condition category: normal nutrient_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. plain_language: At high resolution the acetyl group seals the groove completely, and salicylate is caught sitting where aspirin sits before it reacts. organism: Mouse and human enzyme tissue_or_cell_type: Crystallised cyclooxygenase-2 experimental_model: Crystal structures of an S530T murine COX-2 mutant, aspirin-acetylated human COX-2 and human COX-2 with salicylate, at 1.9 to 2.4 angstrom limitations: Much higher resolution than the 1995 structure and it captures salicylate separately. The authors note that detergent and lipid in the membrane-binding domain change the acetylation rate in vitro, which is a caution about all such measurements. exposure: Aspirin acetylation and salicylate binding, with an S530T/G533V double mutant and purification in several detergent systems evidence_span: {"source_cache": "artifacts/aspirin-research/26859324.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "2d5dab1d6b7576a909c9cef6b5c247e6a399946afbf7ef6d428d67475a363c34", "start_char": 0, "end_char": 1553, "text_sha256": "2d5dab1d6b7576a909c9cef6b5c247e6a399946afbf7ef6d428d67475a363c34"} [asa-p26859324] Crystal Structure of Aspirin-Acetylated Human Cyclooxygenase-2: Insight into the Formation of Products with Reversed Stereochemistry. (2016). https://pubmed.ncbi.nlm.nih.gov/26859324/ DOI: 10.1021/acs.biochem.5b01378

evidence_span

{"source_cache": "artifacts/aspirin-research/2108169.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "c7b11508521a3854b432f3836de6e92ba9863c8edb244d747bb1c37634c0bb50", "start_char": 0, "end_char": 2076, "text_sha256": "c7b11508521a3854b432f3836de6e92ba9863c8edb244d747bb1c37634c0bb50"}

experimental_model

Site-directed mutagenesis of sheep prostaglandin endoperoxide synthase expressed in Cos-1 cells

exposure

Serine 530 replaced with alanine, compared with the native enzyme against aspirin and reversible inhibitors

limitations

The decisive experiment for the mechanism: it separates what the serine does for catalysis from what acetylating it does. Recombinant sheep enzyme, not human enzyme in a tissue.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Sheep and mouse enzyme

plain_language

The acetyl group works by being in the way, not by removing something the enzyme needed.

primary_references

[asa-p2108169] The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. (1990). https://pubmed.ncbi.nlm.nih.gov/2108169/ DOI: 10.1016/s0021-9258(19)34105-5

tissue_or_cell_type

Recombinant enzyme in Cos-1 cells

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 91–102

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Site-directed mutagenesis of sheep prostaglandin endoperoxide synthase expressed in Cos-1 cells · source_derived_draft · unverified_draft

### asa-acetylation-is-steric Acetylation of Ser-530 of sheep prostaglandin endoperoxide synthase by aspirin causes irreversible inactivation of the cyclooxygenase activity of the enzyme, and since the serine hydroxyl is not required for catalysis the acetylation apparently introduces a bulky side chain at position 530 which interferes with arachidonate binding; a sequence of 35 residues with Ser-530 at the midpoint was identical in the sheep and mouse enzymes. Condition category: normal nutrient_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. plain_language: The acetyl group works by being in the way, not by removing something the enzyme needed. organism: Sheep and mouse enzyme tissue_or_cell_type: Recombinant enzyme in Cos-1 cells experimental_model: Site-directed mutagenesis of sheep prostaglandin endoperoxide synthase expressed in Cos-1 cells limitations: The decisive experiment for the mechanism: it separates what the serine does for catalysis from what acetylating it does. Recombinant sheep enzyme, not human enzyme in a tissue. exposure: Serine 530 replaced with alanine, compared with the native enzyme against aspirin and reversible inhibitors evidence_span: {"source_cache": "artifacts/aspirin-research/2108169.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "c7b11508521a3854b432f3836de6e92ba9863c8edb244d747bb1c37634c0bb50", "start_char": 0, "end_char": 2076, "text_sha256": "c7b11508521a3854b432f3836de6e92ba9863c8edb244d747bb1c37634c0bb50"} [asa-p2108169] The aspirin and heme-binding sites of ovine and murine prostaglandin endoperoxide synthases. (1990). https://pubmed.ncbi.nlm.nih.gov/2108169/ DOI: 10.1016/s0021-9258(19)34105-5

evidence_span

{"source_cache": "artifacts/aspirin-research/2144687.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "0a66f17b41129423ecb73bf520dad47b666792a6477552db04272474fc79be24", "start_char": 0, "end_char": 1595, "text_sha256": "0a66f17b41129423ecb73bf520dad47b666792a6477552db04272474fc79be24"}

experimental_model

Sequence comparison and site-directed mutagenesis across sheep, mouse and human prostaglandin endoperoxide synthase

exposure

Ser530 replaced with alanine and with asparagine, and tetranitromethane modification with ibuprofen protection

limitations

Extends the single mutant to a pair that differ in bulk. Sequence-based assignment of the heme ligands is inference from similarity, not structure.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Sheep, mouse and human enzyme

plain_language

Swap in a small residue and the enzyme is fine; swap in a bigger one and the cyclooxygenase half stops.

primary_references

[asa-p2144687] Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases. (1990). https://pubmed.ncbi.nlm.nih.gov/2144687/

tissue_or_cell_type

Recombinant enzyme

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 104–115

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Sequence comparison and site-directed mutagenesis across sheep, mouse and human prostaglandin endoperoxide synthase · source_derived_draft · unverified_draft

### asa-bulk-not-identity The Ala530 mutant of prostaglandin endoperoxide synthase had both cyclooxygenase and hydroperoxidase activity while the Asn530 mutant lacked cyclooxygenase activity but retained hydroperoxidase activity, establishing that the hydroxyl group of Ser530 is not essential for catalysis or substrate binding and suggesting that a bulky group at position 530, such as that introduced by aspirin acetylation, prevents arachidonate binding to the cyclooxygenase active site. Condition category: normal nutrient_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. plain_language: Swap in a small residue and the enzyme is fine; swap in a bigger one and the cyclooxygenase half stops. organism: Sheep, mouse and human enzyme tissue_or_cell_type: Recombinant enzyme experimental_model: Sequence comparison and site-directed mutagenesis across sheep, mouse and human prostaglandin endoperoxide synthase limitations: Extends the single mutant to a pair that differ in bulk. Sequence-based assignment of the heme ligands is inference from similarity, not structure. exposure: Ser530 replaced with alanine and with asparagine, and tetranitromethane modification with ibuprofen protection evidence_span: {"source_cache": "artifacts/aspirin-research/2144687.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "0a66f17b41129423ecb73bf520dad47b666792a6477552db04272474fc79be24", "start_char": 0, "end_char": 1595, "text_sha256": "0a66f17b41129423ecb73bf520dad47b666792a6477552db04272474fc79be24"} [asa-p2144687] Structure-function relationships in sheep, mouse, and human prostaglandin endoperoxide G/H synthases. (1990). https://pubmed.ncbi.nlm.nih.gov/2144687/

evidence_span

{"source_cache": "artifacts/aspirin-research/1601897.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "616cbc367eb38194b858872cbf91108baa3b1dec7aa039a40f69a29fa7157c2d", "start_char": 0, "end_char": 1744, "text_sha256": "616cbc367eb38194b858872cbf91108baa3b1dec7aa039a40f69a29fa7157c2d"}

experimental_model

Five further substitutions at Ser-530 and substitutions at adjoining residues, expressed transiently in cos-1 cells

exposure

A graded series of side chains at position 530, plus substitutions at Phe-529, Leu-531, Lys-532 and Gly-533

limitations

Tests the steric hypothesis by varying the bulk systematically rather than with one mutant. Transient expression in a heterologous cell line.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Enzyme

plain_language

The bigger the group put at that position, the worse the enzyme binds its substrate.

primary_references

[asa-p1601897] Prostaglandin endoperoxide synthase. The aspirin acetylation region. (1992). https://pubmed.ncbi.nlm.nih.gov/1601897/ DOI: 10.1016/s0021-9258(19)49852-9

tissue_or_cell_type

Recombinant prostaglandin endoperoxide H synthase-1

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 117–128

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · Five further substitutions at Ser-530 and substitutions at adjoining residues, expressed transiently in cos-1 cells · source_derived_draft · unverified_draft

### asa-graded-bulk-series Using site-directed mutagenesis to prepare five further substitutions of Ser-530, the presence of amino acids with bulky side chains at position 530 inhibited cyclooxygenase activity and decreased the apparent affinity of the enzyme for arachidonate, consistent with the proposal that aspirin inhibits by placing a larger than normal side chain at position 530; residues 529 to 533 all proved important for the peroxidase as well as the cyclooxygenase activity, and Phe-529 in particular was critical for structure and catalysis. Condition category: normal nutrient_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. plain_language: The bigger the group put at that position, the worse the enzyme binds its substrate. organism: Enzyme tissue_or_cell_type: Recombinant prostaglandin endoperoxide H synthase-1 experimental_model: Five further substitutions at Ser-530 and substitutions at adjoining residues, expressed transiently in cos-1 cells limitations: Tests the steric hypothesis by varying the bulk systematically rather than with one mutant. Transient expression in a heterologous cell line. exposure: A graded series of side chains at position 530, plus substitutions at Phe-529, Leu-531, Lys-532 and Gly-533 evidence_span: {"source_cache": "artifacts/aspirin-research/1601897.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "616cbc367eb38194b858872cbf91108baa3b1dec7aa039a40f69a29fa7157c2d", "start_char": 0, "end_char": 1744, "text_sha256": "616cbc367eb38194b858872cbf91108baa3b1dec7aa039a40f69a29fa7157c2d"} [asa-p1601897] Prostaglandin endoperoxide synthase. The aspirin acetylation region. (1992). https://pubmed.ncbi.nlm.nih.gov/1601897/ DOI: 10.1016/s0021-9258(19)49852-9

evidence_span

{"source_cache": "artifacts/aspirin-research/7552725.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "dd61431f20eb312313942bc8a89f970d5d2e33600b32abe692f20d9d5c57d7b1", "start_char": 0, "end_char": 751, "text_sha256": "dd61431f20eb312313942bc8a89f970d5d2e33600b32abe692f20d9d5c57d7b1"}

experimental_model

X-ray crystal structure at 3.4 angstrom of prostaglandin H2 synthase-1 inactivated by a potent aspirin analogue

exposure

2-bromoacetoxy-benzoic acid acetylation, with salicylic acid soaked into the structure

limitations

Direct structural evidence rather than inference from mutants, though at modest resolution and using an analogue rather than aspirin itself.

nutrient_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. · Aspirin / acetylsalicylic acid

organism

Enzyme

plain_language

The picture confirms it: the acetyl group simply plugs the tunnel the fatty acid has to travel down.

primary_references

[asa-p7552725] The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase. (1995). https://pubmed.ncbi.nlm.nih.gov/7552725/ DOI: 10.1038/nsb0895-637

tissue_or_cell_type

Crystallised prostaglandin H2 synthase-1

Aspirin: the serine it acetylates, the enzyme that acetylation creates, the dose that separates platelet from vessel wall, and the metabolite that is a different drug (2026-09-22) · lines 130–141

AI-assisted literature curation; primary study URLs and scope retained in the document and extraction. Not publisher full text. · supports · X-ray crystal structure at 3.4 angstrom of prostaglandin H2 synthase-1 inactivated by a potent aspirin analogue · source_derived_draft · unverified_draft

### asa-structure-shows-blockage The 3.4 angstrom X-ray crystal structure of prostaglandin H2 synthase isoform-1 inactivated by the potent aspirin analogue 2-bromoacetoxy-benzoic acid shows that acetylation abolishes cyclooxygenase activity by steric blockage of the active-site channel and not through a large conformational change, with two rotameric states of the acetyl-serine side chain blocking the channel to different extents, a result which may explain the dissimilar effects of aspirin on the two isoforms. Condition category: normal nutrient_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. plain_language: The picture confirms it: the acetyl group simply plugs the tunnel the fatty acid has to travel down. organism: Enzyme tissue_or_cell_type: Crystallised prostaglandin H2 synthase-1 experimental_model: X-ray crystal structure at 3.4 angstrom of prostaglandin H2 synthase-1 inactivated by a potent aspirin analogue limitations: Direct structural evidence rather than inference from mutants, though at modest resolution and using an analogue rather than aspirin itself. exposure: 2-bromoacetoxy-benzoic acid acetylation, with salicylic acid soaked into the structure evidence_span: {"source_cache": "artifacts/aspirin-research/7552725.abstract.txt", "locator": "Indexed abstract; zero-based, end-exclusive Unicode character offsets", "file_sha256": "dd61431f20eb312313942bc8a89f970d5d2e33600b32abe692f20d9d5c57d7b1", "start_char": 0, "end_char": 751, "text_sha256": "dd61431f20eb312313942bc8a89f970d5d2e33600b32abe692f20d9d5c57d7b1"} [asa-p7552725] The structural basis of aspirin activity inferred from the crystal structure of inactivated prostaglandin H2 synthase. (1995). https://pubmed.ncbi.nlm.nih.gov/7552725/ DOI: 10.1038/nsb0895-637