{"gene":"PF3D7_0626800","run_date":"2026-09-08T12:51:26","timeline":{"discoveries":[{"year":2005,"finding":"PyrK (PF3D7_0626800, designated PK1 in this study) was cloned from P. falciparum intraerythrocytic-stage mRNA, expressed as a recombinant protein (55.6 kDa, pI 7.5), and shown to be enzymatically active as a homotetramer. It exhibits hyperbolic kinetics for phosphoenolpyruvate (Km = 0.19 mM) and ADP (Km = 0.12 mM). Unlike most pyruvate kinases, it is not activated by fructose-1,6-bisphosphate or glucose-6-phosphate. It is inhibited by ATP (competitive with PEP) and citrate (non-competitive with ADP, Ki = 0.8 mM); citrate shifts PEP kinetics from hyperbolic to sigmoidal. It is partially inactivated by pyridoxal-5'-phosphate.","method":"Recombinant protein expression, in vitro enzymatic assay, kinetic analysis, inhibitor screening","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro enzymatic characterization with multiple substrates, inhibitors, and kinetic parameters; single lab but multiple orthogonal biochemical methods","pmids":["15567170"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"The first crystal structure of PyrK (PF3D7_0626800, referred to as PfPYK) was solved in complex with substrate analogues oxalate and the ATP product. Structural comparison of the active R-state and inactive T-state reveals a 'rock-and-lock' allosteric mechanism driven by rigid-body rotations of each subunit in the tetramer. Kinetic data show glucose-6-phosphate acts as an activator by increasing the apparent maximal velocity of the enzyme. The trypanosome drug suramin was found to inhibit PyrK activity.","method":"X-ray crystallography, kinetic assays, inhibitor assay","journal":"Biochemical and biophysical research communications","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure with functional kinetic validation, multiple orthogonal methods in a single study","pmids":["32878705"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"finding":"Cysteine residues C49 and C343 of PyrK (PF3D7_0626800, referred to as PfPK) are essential for structural and functional integrity. Mutation of either residue (C49A or C343A) resulted in altered substrate affinity, reduced enzymatic activity, and conformational changes within the A-domain (substrate binding site) as determined by X-ray crystallography. The C49A mutant showed more severe effects, with loss of two hydrogen bonds causing multiple conformational changes that impeded proper substrate binding and phosphate transfer.","method":"Site-directed mutagenesis, in vitro enzymatic assay, X-ray crystallography","journal":"Structure","confidence":"High","confidence_rationale":"Tier 1 / Moderate — mutagenesis combined with crystallographic structural validation and enzymatic activity measurement; single lab with multiple orthogonal methods","pmids":["35998635"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"finding":"PyrK (PF3D7_0626800, referred to as PK) is a target of S-glutathionylation in P. falciparum, and this modification reversibly inhibits its enzymatic activity. Deglutathionylation is catalyzed by P. falciparum glutaredoxin 1, thioredoxin 1, and plasmoredoxin.","method":"Enzymatic deglutathionylation, affinity purification, proteomic identification, activity assay","journal":"Antioxidants & redox signaling","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — biochemical activity assay demonstrating reversible inhibition plus identification of deglutathionylating enzymes; single lab","pmids":["21595565"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"Both PK1 (PyrK, PF3D7_0626800) and PK2 are actively transcribed during the intraerythrocytic stages of P. falciparum, as demonstrated by RT-PCR. Amino acid comparison of effector binding sites of PK1 and PK2 to human pyruvate kinases revealed significant differences. Phylogenetic analysis showed the PK2 type is confined to Apicomplexans.","method":"RT-PCR, bioinformatic sequence/phylogenetic analysis","journal":"Travel medicine and infectious disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — RT-PCR transcript detection only; no protein-level functional characterization of PyrK beyond transcription","pmids":["17298920"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2009,"finding":"In pyruvate kinase-deficient (PKD) erythrocytes infected with P. falciparum ring-stage parasites, parasite-derived pyruvate kinase (PyrK, PF3D7_0626800) is up-regulated in response to low intraerythrocytic ATP levels, as indicated by increased P. falciparum-specific pyruvate kinase activity measured in parasitized PKD erythrocytes.","method":"Enzymatic activity assay in parasitized erythrocytes from PKD donors versus controls","journal":"The Journal of infectious diseases","confidence":"Low","confidence_rationale":"Tier 3 / Weak — activity assay in a mixed cellular system without direct molecular identification of parasite-specific enzyme; single lab, indirect evidence of up-regulation","pmids":["19743919"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1988,"finding":"PyrK (pyruvate kinase) activity in P. falciparum-infected erythrocytes is extremely elevated compared to uninfected red cells, and electrophoretically distinct bands of pyruvate kinase activity were detected in infected cells, suggesting expression of a parasite-specific enzyme isoform.","method":"Enzymatic activity assay, gel electrophoresis of enzyme activity","journal":"Blood","confidence":"Low","confidence_rationale":"Tier 3 / Weak — indirect biochemical evidence from mixed cell populations; no molecular characterization of the parasite enzyme itself","pmids":["3058230"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"finding":"Conditioned culture medium from P. falciparum-infected red blood cells directly inhibits pyruvate kinase (PK) activity in uninfected RBCs, suggesting that a secreted parasite factor can modulate PK activity. This inhibition was specific to regulatory glycolytic enzymes (PFK and PK) and did not affect G-6-PD.","method":"Enzymatic activity assay in RBCs treated with conditioned medium","journal":"Journal of vector borne diseases","confidence":"Low","confidence_rationale":"Tier 3 / Weak — enzymatic assay in a mixed/indirect system, inhibition attributed to conditioned medium without molecular identification of the inhibitory factor; single study","pmids":["17024857"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2026,"finding":"Acyl carrier protein (ACP) in the P. falciparum apicoplast was found to bind and stabilize apicoplast pyruvate kinase II (PKII, PyrKII; a distinct isoform from PyrK/PF3D7_0626800). Loss of ACP leads to destabilization and loss of PKII, which is sufficient to explain ACP essentiality in blood-stage parasites. This finding pertains to PyrKII (the apicoplast isoform), NOT PyrK (PF3D7_0626800).","method":"Proximity biotinylation, biochemical interaction studies, conditional knockdown","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 2 / Weak — finding is about the PyrKII isoform (apicoplast-localized), not PyrK/PF3D7_0626800; included here for completeness but not attributed to the query gene","pmids":["41959251"],"is_preprint":true,"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"current_model":"PyrK (PF3D7_0626800) is a cytoplasmic glycolytic pyruvate kinase expressed during P. falciparum intraerythrocytic stages that functions as a homotetramer catalyzing phosphoenolpyruvate (Km 0.19 mM) and ADP (Km 0.12 mM) with hyperbolic kinetics; its activity is allosterically regulated via a 'rock-and-lock' R/T-state mechanism (defined by crystal structure), activated by glucose-6-phosphate, inhibited by ATP and citrate, reversibly inhibited by S-glutathionylation (reversed by glutaredoxin 1, thioredoxin 1, and plasmoredoxin), and critically dependent on cysteine residues C49 and C343 for substrate binding and catalytic integrity."},"narrative":{"mechanistic_narrative":"PyrK (PF3D7_0626800) is a glycolytic pyruvate kinase expressed during the intraerythrocytic stages of Plasmodium falciparum that catalyzes the phosphoenolpyruvate/ADP reaction as an active homotetramer with hyperbolic kinetics (PEP Km = 0.19 mM, ADP Km = 0.12 mM) [PMID:15567170]. Its allosteric regulation departs from canonical human pyruvate kinases: it is activated by glucose-6-phosphate, which raises apparent maximal velocity, and is inhibited by ATP (competitive with PEP) and citrate (non-competitive, shifting PEP kinetics from hyperbolic to sigmoidal) [PMID:15567170, PMID:32878705]. The active R-state and inactive T-state crystal structures define a 'rock-and-lock' mechanism in which rigid-body rotation of each tetramer subunit gates activity [PMID:32878705]. Substrate binding and catalytic integrity depend on the A-domain cysteines C49 and C343, whose mutation causes conformational changes, altered substrate affinity, and reduced activity [PMID:35998635]. PyrK is also subject to reversible redox control through S-glutathionylation, which inhibits the enzyme and is reversed by P. falciparum glutaredoxin 1, thioredoxin 1, and plasmoredoxin [PMID:21595565]. The trypanosome drug suramin inhibits PyrK, and effector-binding-site divergence from human isoforms marks it as a potential antimalarial target [PMID:32878705, PMID:17298920]. No physical partners or complex membership of PyrK itself have been characterized in the available corpus beyond its enzymatic deglutathionylation by thioredoxin-family enzymes.","teleology":[{"year":1988,"claim":"Established that P. falciparum infection introduces a parasite-specific pyruvate kinase activity into the host erythrocyte, motivating molecular characterization of a distinct parasite enzyme.","evidence":"Enzymatic activity assay and gel electrophoresis of PK activity in infected versus uninfected red cells","pmids":["3058230"],"confidence":"Low","gaps":["Indirect evidence from mixed cell populations; the parasite enzyme was not molecularly identified","Cannot distinguish PyrK from other pyruvate kinase isoforms"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2005,"claim":"Answered what the recombinant parasite enzyme is and how it behaves kinetically, showing PyrK is an active homotetramer with an unusual regulatory profile distinct from most pyruvate kinases.","evidence":"Recombinant expression, in vitro kinetic and inhibitor assays","pmids":["15567170"],"confidence":"High","gaps":["Regulation reported here as insensitive to G6P, later refined by structural study","No structural basis for allostery yet","Cellular/in vivo role not addressed"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2006,"claim":"Confirmed PyrK is actively transcribed during blood stages and that its effector-binding sites diverge from human pyruvate kinases, framing it as a candidate drug target.","evidence":"RT-PCR transcript detection and bioinformatic sequence/phylogenetic analysis","pmids":["17298920"],"confidence":"Low","gaps":["Transcript detection only, no protein-level functional data in this study","Does not establish differential druggability experimentally"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2011,"claim":"Revealed a redox regulatory layer, showing PyrK activity is reversibly inhibited by S-glutathionylation and restored by parasite thioredoxin-family enzymes.","evidence":"Enzymatic deglutathionylation assays with glutaredoxin 1, thioredoxin 1, and plasmoredoxin; proteomic identification","pmids":["21595565"],"confidence":"Medium","gaps":["Specific modified cysteines not mapped in this study","Physiological trigger and in vivo relevance of the modification unresolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"claim":"Provided the structural basis for allostery, defining a 'rock-and-lock' R/T-state mechanism and identifying G6P as an activator and suramin as an inhibitor.","evidence":"X-ray crystallography with oxalate/ATP ligands, kinetic and inhibitor assays","pmids":["32878705"],"confidence":"High","gaps":["Suramin selectivity over human enzymes and in-cell efficacy not established","Physiological signal driving R/T transition in the parasite unknown"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2022,"claim":"Pinpointed the molecular determinants of catalytic integrity, showing A-domain cysteines C49 and C343 are required for proper substrate binding and phosphate transfer.","evidence":"Site-directed mutagenesis (C49A, C343A), enzymatic assays, and X-ray crystallography of mutants","pmids":["35998635"],"confidence":"High","gaps":["Does not directly link these cysteines to the S-glutathionylation site","No cellular validation of mutant phenotypes in parasites"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":null,"claim":"Whether PyrK is essential for blood-stage growth in vivo, its physical partners, and whether suramin or cysteine-directed inhibitors can be developed into selective antimalarials remain unaddressed in the available corpus.","evidence":"","pmids":[],"confidence":"Low","gaps":["No genetic knockout/knockdown of PyrK reported","No interaction partners or complex membership established for PyrK itself","Druggability of PyrK-selective inhibitors not tested in parasites"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Homotetramer [#0]"],"partners":["GLUTAREDOXIN 1 (DEGLUTATHIONYLATION OF PYRK) [#3]","THIOREDOXIN 1 (DEGLUTATHIONYLATION OF PYRK) [#3]","PLASMOREDOXIN (DEGLUTATHIONYLATION OF PYRK) [#3]"],"other_free_text":["Activated by glucose-6-phosphate (increases apparent Vmax); inhibited by ATP (competitive with PEP) and citrate (non-competitive, induces sigmoidal PEP kinetics) [#0, #1]","Allosteric 'rock-and-lock' R/T-state mechanism defined by crystallography [#1]","Catalytic A-domain cysteines C49 and C343 essential for substrate binding and phosphate transfer [#2]","Reversibly inhibited by S-glutathionylation [#3]","Inhibited by suramin; partially inactivated by pyridoxal-5'-phosphate [#0, #1]","Effector-binding sites diverge from human pyruvate kinases [#4]","Note: discovery [#8] concerns the apicoplast isoform PyrKII and its ACP interaction, NOT PyrK/PF3D7_0626800; not attributed to the query gene"]}},"prefetch_data":{},"corpus_meta":[{"pmid":"3058230","id":"PMC_3058230","title":"The enzymes of the glycolytic pathway in erythrocytes infected with Plasmodium falciparum malaria parasites.","date":"1988","source":"Blood","url":"https://pubmed.ncbi.nlm.nih.gov/3058230","citation_count":98,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["pyruvate kinase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false},{"pmid":"2257322","id":"PMC_2257322","title":"Plasmodium falciparum carbohydrate metabolism: a connection between host cell and parasite.","date":"1990","source":"Blood cells","url":"https://pubmed.ncbi.nlm.nih.gov/2257322","citation_count":95,"is_preprint":false,"source_species":["Pf"],"role":"primary","match_terms":["pyruvate 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kinase"],"competing_organism_flag":false,"competing_organism_match":"","competing_organism_only":false}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":14705,"output_tokens":2729,"usd":0.042525,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":8272,"output_tokens":2960,"usd":0.05768,"stage2_stop_reason":"end_turn"},"total_usd":0.100205,"stage1_batch_id":"msgbatch_01DisAaTypwmC8QwdMBuePHo","stage2_batch_id":"msgbatch_0184zxZTqxiEfdYYfPMfkqjb","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"PyrK (PF3D7_0626800, designated PK1 in this study) was cloned from P. falciparum intraerythrocytic-stage mRNA, expressed as a recombinant protein (55.6 kDa, pI 7.5), and shown to be enzymatically active as a homotetramer. It exhibits hyperbolic kinetics for phosphoenolpyruvate (Km = 0.19 mM) and ADP (Km = 0.12 mM). Unlike most pyruvate kinases, it is not activated by fructose-1,6-bisphosphate or glucose-6-phosphate. It is inhibited by ATP (competitive with PEP) and citrate (non-competitive with ADP, Ki = 0.8 mM); citrate shifts PEP kinetics from hyperbolic to sigmoidal. It is partially inactivated by pyridoxal-5'-phosphate.\",\n      \"method\": \"Recombinant protein expression, in vitro enzymatic assay, kinetic analysis, inhibitor screening\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro enzymatic characterization with multiple substrates, inhibitors, and kinetic parameters; single lab but multiple orthogonal biochemical methods\",\n      \"pmids\": [\"15567170\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The first crystal structure of PyrK (PF3D7_0626800, referred to as PfPYK) was solved in complex with substrate analogues oxalate and the ATP product. Structural comparison of the active R-state and inactive T-state reveals a 'rock-and-lock' allosteric mechanism driven by rigid-body rotations of each subunit in the tetramer. Kinetic data show glucose-6-phosphate acts as an activator by increasing the apparent maximal velocity of the enzyme. The trypanosome drug suramin was found to inhibit PyrK activity.\",\n      \"method\": \"X-ray crystallography, kinetic assays, inhibitor assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure with functional kinetic validation, multiple orthogonal methods in a single study\",\n      \"pmids\": [\"32878705\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Cysteine residues C49 and C343 of PyrK (PF3D7_0626800, referred to as PfPK) are essential for structural and functional integrity. Mutation of either residue (C49A or C343A) resulted in altered substrate affinity, reduced enzymatic activity, and conformational changes within the A-domain (substrate binding site) as determined by X-ray crystallography. The C49A mutant showed more severe effects, with loss of two hydrogen bonds causing multiple conformational changes that impeded proper substrate binding and phosphate transfer.\",\n      \"method\": \"Site-directed mutagenesis, in vitro enzymatic assay, X-ray crystallography\",\n      \"journal\": \"Structure\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — mutagenesis combined with crystallographic structural validation and enzymatic activity measurement; single lab with multiple orthogonal methods\",\n      \"pmids\": [\"35998635\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"PyrK (PF3D7_0626800, referred to as PK) is a target of S-glutathionylation in P. falciparum, and this modification reversibly inhibits its enzymatic activity. Deglutathionylation is catalyzed by P. falciparum glutaredoxin 1, thioredoxin 1, and plasmoredoxin.\",\n      \"method\": \"Enzymatic deglutathionylation, affinity purification, proteomic identification, activity assay\",\n      \"journal\": \"Antioxidants & redox signaling\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — biochemical activity assay demonstrating reversible inhibition plus identification of deglutathionylating enzymes; single lab\",\n      \"pmids\": [\"21595565\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Both PK1 (PyrK, PF3D7_0626800) and PK2 are actively transcribed during the intraerythrocytic stages of P. falciparum, as demonstrated by RT-PCR. Amino acid comparison of effector binding sites of PK1 and PK2 to human pyruvate kinases revealed significant differences. Phylogenetic analysis showed the PK2 type is confined to Apicomplexans.\",\n      \"method\": \"RT-PCR, bioinformatic sequence/phylogenetic analysis\",\n      \"journal\": \"Travel medicine and infectious disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — RT-PCR transcript detection only; no protein-level functional characterization of PyrK beyond transcription\",\n      \"pmids\": [\"17298920\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"In pyruvate kinase-deficient (PKD) erythrocytes infected with P. falciparum ring-stage parasites, parasite-derived pyruvate kinase (PyrK, PF3D7_0626800) is up-regulated in response to low intraerythrocytic ATP levels, as indicated by increased P. falciparum-specific pyruvate kinase activity measured in parasitized PKD erythrocytes.\",\n      \"method\": \"Enzymatic activity assay in parasitized erythrocytes from PKD donors versus controls\",\n      \"journal\": \"The Journal of infectious diseases\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — activity assay in a mixed cellular system without direct molecular identification of parasite-specific enzyme; single lab, indirect evidence of up-regulation\",\n      \"pmids\": [\"19743919\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"PyrK (pyruvate kinase) activity in P. falciparum-infected erythrocytes is extremely elevated compared to uninfected red cells, and electrophoretically distinct bands of pyruvate kinase activity were detected in infected cells, suggesting expression of a parasite-specific enzyme isoform.\",\n      \"method\": \"Enzymatic activity assay, gel electrophoresis of enzyme activity\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — indirect biochemical evidence from mixed cell populations; no molecular characterization of the parasite enzyme itself\",\n      \"pmids\": [\"3058230\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"Conditioned culture medium from P. falciparum-infected red blood cells directly inhibits pyruvate kinase (PK) activity in uninfected RBCs, suggesting that a secreted parasite factor can modulate PK activity. This inhibition was specific to regulatory glycolytic enzymes (PFK and PK) and did not affect G-6-PD.\",\n      \"method\": \"Enzymatic activity assay in RBCs treated with conditioned medium\",\n      \"journal\": \"Journal of vector borne diseases\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — enzymatic assay in a mixed/indirect system, inhibition attributed to conditioned medium without molecular identification of the inhibitory factor; single study\",\n      \"pmids\": [\"17024857\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2026,\n      \"finding\": \"Acyl carrier protein (ACP) in the P. falciparum apicoplast was found to bind and stabilize apicoplast pyruvate kinase II (PKII, PyrKII; a distinct isoform from PyrK/PF3D7_0626800). Loss of ACP leads to destabilization and loss of PKII, which is sufficient to explain ACP essentiality in blood-stage parasites. This finding pertains to PyrKII (the apicoplast isoform), NOT PyrK (PF3D7_0626800).\",\n      \"method\": \"Proximity biotinylation, biochemical interaction studies, conditional knockdown\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 2 / Weak — finding is about the PyrKII isoform (apicoplast-localized), not PyrK/PF3D7_0626800; included here for completeness but not attributed to the query gene\",\n      \"pmids\": [\"41959251\"],\n      \"is_preprint\": true,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"current_model\": \"PyrK (PF3D7_0626800) is a cytoplasmic glycolytic pyruvate kinase expressed during P. falciparum intraerythrocytic stages that functions as a homotetramer catalyzing phosphoenolpyruvate (Km 0.19 mM) and ADP (Km 0.12 mM) with hyperbolic kinetics; its activity is allosterically regulated via a 'rock-and-lock' R/T-state mechanism (defined by crystal structure), activated by glucose-6-phosphate, inhibited by ATP and citrate, reversibly inhibited by S-glutathionylation (reversed by glutaredoxin 1, thioredoxin 1, and plasmoredoxin), and critically dependent on cysteine residues C49 and C343 for substrate binding and catalytic integrity.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PyrK (PF3D7_0626800) is a glycolytic pyruvate kinase expressed during the intraerythrocytic stages of Plasmodium falciparum that catalyzes the phosphoenolpyruvate/ADP reaction as an active homotetramer with hyperbolic kinetics (PEP Km = 0.19 mM, ADP Km = 0.12 mM) [#0]. Its allosteric regulation departs from canonical human pyruvate kinases: it is activated by glucose-6-phosphate, which raises apparent maximal velocity, and is inhibited by ATP (competitive with PEP) and citrate (non-competitive, shifting PEP kinetics from hyperbolic to sigmoidal) [#0, #1]. The active R-state and inactive T-state crystal structures define a 'rock-and-lock' mechanism in which rigid-body rotation of each tetramer subunit gates activity [#1]. Substrate binding and catalytic integrity depend on the A-domain cysteines C49 and C343, whose mutation causes conformational changes, altered substrate affinity, and reduced activity [#2]. PyrK is also subject to reversible redox control through S-glutathionylation, which inhibits the enzyme and is reversed by P. falciparum glutaredoxin 1, thioredoxin 1, and plasmoredoxin [#3]. The trypanosome drug suramin inhibits PyrK, and effector-binding-site divergence from human isoforms marks it as a potential antimalarial target [#1, #4]. No physical partners or complex membership of PyrK itself have been characterized in the available corpus beyond its enzymatic deglutathionylation by thioredoxin-family enzymes.\",\n  \"teleology\": [\n    {\n      \"year\": 1988,\n      \"claim\": \"Established that P. falciparum infection introduces a parasite-specific pyruvate kinase activity into the host erythrocyte, motivating molecular characterization of a distinct parasite enzyme.\",\n      \"evidence\": \"Enzymatic activity assay and gel electrophoresis of PK activity in infected versus uninfected red cells\",\n      \"pmids\": [\"3058230\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Indirect evidence from mixed cell populations; the parasite enzyme was not molecularly identified\", \"Cannot distinguish PyrK from other pyruvate kinase isoforms\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Answered what the recombinant parasite enzyme is and how it behaves kinetically, showing PyrK is an active homotetramer with an unusual regulatory profile distinct from most pyruvate kinases.\",\n      \"evidence\": \"Recombinant expression, in vitro kinetic and inhibitor assays\",\n      \"pmids\": [\"15567170\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Regulation reported here as insensitive to G6P, later refined by structural study\", \"No structural basis for allostery yet\", \"Cellular/in vivo role not addressed\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Confirmed PyrK is actively transcribed during blood stages and that its effector-binding sites diverge from human pyruvate kinases, framing it as a candidate drug target.\",\n      \"evidence\": \"RT-PCR transcript detection and bioinformatic sequence/phylogenetic analysis\",\n      \"pmids\": [\"17298920\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Transcript detection only, no protein-level functional data in this study\", \"Does not establish differential druggability experimentally\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Revealed a redox regulatory layer, showing PyrK activity is reversibly inhibited by S-glutathionylation and restored by parasite thioredoxin-family enzymes.\",\n      \"evidence\": \"Enzymatic deglutathionylation assays with glutaredoxin 1, thioredoxin 1, and plasmoredoxin; proteomic identification\",\n      \"pmids\": [\"21595565\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific modified cysteines not mapped in this study\", \"Physiological trigger and in vivo relevance of the modification unresolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Provided the structural basis for allostery, defining a 'rock-and-lock' R/T-state mechanism and identifying G6P as an activator and suramin as an inhibitor.\",\n      \"evidence\": \"X-ray crystallography with oxalate/ATP ligands, kinetic and inhibitor assays\",\n      \"pmids\": [\"32878705\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Suramin selectivity over human enzymes and in-cell efficacy not established\", \"Physiological signal driving R/T transition in the parasite unknown\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Pinpointed the molecular determinants of catalytic integrity, showing A-domain cysteines C49 and C343 are required for proper substrate binding and phosphate transfer.\",\n      \"evidence\": \"Site-directed mutagenesis (C49A, C343A), enzymatic assays, and X-ray crystallography of mutants\",\n      \"pmids\": [\"35998635\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not directly link these cysteines to the S-glutathionylation site\", \"No cellular validation of mutant phenotypes in parasites\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether PyrK is essential for blood-stage growth in vivo, its physical partners, and whether suramin or cysteine-directed inhibitors can be developed into selective antimalarials remain unaddressed in the available corpus.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No genetic knockout/knockdown of PyrK reported\", \"No interaction partners or complex membership established for PyrK itself\", \"Druggability of PyrK-selective inhibitors not tested in parasites\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004743\", \"supporting_discovery_ids\": [0, 1, 2]},\n      {\"term_id\": \"GO:0000287\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006096\", \"supporting_discovery_ids\": [0, 1]}\n    ],\n    \"complexes\": [\n      \"Homotetramer [#0]\"\n    ],\n    \"partners\": [\n      \"Glutaredoxin 1 (deglutathionylation of PyrK) [#3]\",\n      \"Thioredoxin 1 (deglutathionylation of PyrK) [#3]\",\n      \"Plasmoredoxin (deglutathionylation of PyrK) [#3]\"\n    ],\n    \"other_free_text\": [\n      \"Activated by glucose-6-phosphate (increases apparent Vmax); inhibited by ATP (competitive with PEP) and citrate (non-competitive, induces sigmoidal PEP kinetics) [#0, #1]\",\n      \"Allosteric 'rock-and-lock' R/T-state mechanism defined by crystallography [#1]\",\n      \"Catalytic A-domain cysteines C49 and C343 essential for substrate binding and phosphate transfer [#2]\",\n      \"Reversibly inhibited by S-glutathionylation [#3]\",\n      \"Inhibited by suramin; partially inactivated by pyridoxal-5'-phosphate [#0, #1]\",\n      \"Effector-binding sites diverge from human pyruvate kinases [#4]\",\n      \"Note: discovery [#8] concerns the apicoplast isoform PyrKII and its ACP interaction, NOT PyrK/PF3D7_0626800; not attributed to the query gene\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0626800","name":null,"product":"pyruvate kinase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1125600","name":null,"product":"pyruvate kinase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1123000","name":null,"product":"pyruvate kinase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"PyrK","evaluation":null}