{"gene":"PF3D7_0624000","run_date":"2026-09-08T12:51:26","timeline":{"discoveries":[{"year":1987,"finding":"P. falciparum HK (PfHK) has distinct kinetic properties from the human erythrocyte enzyme: Km for glucose is 431 µM (vs. 98 µM for the erythrocyte enzyme), while Km for ATP and Vmax for both glucose and ATP are similar. PfHK activity is ~25-fold higher in infected erythrocytes than in control uninfected erythrocytes. The rate of GSSG reduction in infected erythrocyte lysates was proportional to hexokinase activity, establishing that PfHK-driven glucose-6-phosphate production feeds the pentose shunt to generate NADPH for glutathione reduction.","method":"Enzyme kinetics assays on parasite lysates; GSSG reduction assays correlated with parasitemia and hexokinase activity; comparison with G6PD-deficient infected red cell lysates","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzyme kinetics with functional consequence (GSSG reduction), single lab, two orthogonal approaches (kinetics + GSSG reduction assay)","pmids":["3316204"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1988,"finding":"Hexokinase activity is extremely elevated in P. falciparum-infected erythrocytes compared to uninfected red cells, and an electrophoretically distinct band of hexokinase activity is present, indicating parasite-encoded HK distinct from the host enzyme.","method":"Enzyme activity assays and electrophoretic analysis of glycolytic enzymes in infected vs. uninfected erythrocytes and isolated parasites","journal":"Blood","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — electrophoretic separation plus enzyme activity assays, single lab, two orthogonal methods","pmids":["3058230"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1992,"finding":"PfHK is encoded by a single-copy gene on chromosome 8, translated from a single 1.5-kb exon within a 3-kb mRNA during asexual stages, and the protein has ~26% amino acid identity to human hexokinase I. Polyclonal antibodies against recombinant PfHK partially inhibited hexokinase activity in a P. falciparum lysate, confirming the cloned gene encodes the parasite hexokinase.","method":"cDNA/genomic DNA cloning, sequence analysis, Western blotting with poly- and monoclonal antibodies, antibody inhibition of enzyme activity","journal":"Molecular and biochemical parasitology","confidence":"High","confidence_rationale":"Tier 1–2 / Strong — gene cloning, protein-level confirmation by Western blot, functional confirmation by antibody inhibition of enzymatic activity, multiple orthogonal methods","pmids":["1475005"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1994,"finding":"PfHK expression and cellular localization were characterized during blood-stage development of P. falciparum.","method":"Not fully specified in available abstract (cellular localization study)","journal":"Molecular and biochemical parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — abstract not retrievable; only title available, insufficient detail to assess methods rigorously","pmids":["8183319"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1957,"finding":"Hexokinase activity in hemolysates of P. berghei-infected mouse/rat erythrocytes is much higher than in normal erythrocytes, confirming the presence of a parasite hexokinase. Several antimalarial compounds (but not proguanil itself; its metabolite 10,580 was effective) inhibit P. berghei hexokinase in a manner similar to yeast hexokinase. The inhibition by mepacrine was reduced when ATP concentration was raised, while inhibition by compound 10,732 was independent of ATP concentration.","method":"Enzyme inhibition assays on hemolysates of P. berghei-infected rodent erythrocytes; comparison with yeast hexokinase","journal":"British journal of pharmacology and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — enzyme inhibition assays with multiple compounds and ATP competition analysis, single lab","pmids":["13413145"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":1979,"finding":"Parasite-specific hexokinase activity was demonstrated in P. berghei parasites isolated from infected mouse erythrocytes, providing an explanation for increased glucose consumption in infected cells.","method":"Enzyme assays on parasites isolated by hemolysin lysis; serology to confirm purity","journal":"The Journal of parasitology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single enzyme assay, single lab, no mechanistic detail beyond detection","pmids":["376819"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":1990,"finding":"In P. berghei, hexokinase was partially purified from isolated parasites and kinetically characterized; its kinetic properties differ significantly from erythrocyte hexokinase, and it is less efficiently inhibited. The enzyme is present in the cytosolic fraction of the isolated parasite.","method":"Partial purification, enzyme kinetics, subcellular fractionation","journal":"Biomedica biochimica acta","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — partial purification plus kinetic characterization and fractionation, single lab","pmids":["2143651"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":1997,"finding":"P. berghei hexokinase was purified from isolated parasites; its activity is 35-fold higher than in normal mouse erythrocytes and 5-fold higher than in P. berghei-infected erythrocytes. The enzyme has a subunit molecular weight of ~47 kDa and Km for ATP of 2 mM. Maximal activity is present in the cytosolic fraction.","method":"Purification (Sephadex G-200), SDS-PAGE, enzyme kinetics, subcellular fractionation","journal":"Acta veterinaria Hungarica","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — enzyme purification with kinetic characterization and fractionation, single lab","pmids":["9270135"],"is_preprint":false,"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":2008,"finding":"2-fluoro-2-deoxy-D-glucose (2-FG) and 2-deoxy-D-glucose (2-DG) inhibit glucose phosphorylation by PfHK; a trend was observed between antiplasmodial activity of glucose analogs and their ability to inhibit hexokinase-mediated glucose phosphorylation. This validates PfHK and early glycolytic steps as antiplasmodial drug targets.","method":"Hexokinase inhibition assays with glucose analogs; glucose accumulation assays in intraerythrocytic parasites; correlation with antiplasmodial activity","journal":"The Journal of pharmacology and experimental therapeutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro enzyme inhibition plus cellular glucose phosphorylation assay, single lab, two orthogonal approaches","pmids":["18713952"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"finding":"PfHK was captured as a target protein of cytosolic thioredoxin-1 (PfTrx-1) using a cysteine mutant (C33S) Trx-affinity chromatography approach, identifying PfHK as a potential redox-regulated glycolytic enzyme.","method":"Trx-affinity chromatography with a cysteine mutant (mixed disulfide trapping), LC-MS protein identification","journal":"Parasitology international","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single affinity pulldown with MS identification, no direct functional follow-up on PfHK specifically","pmids":["20307687"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"finding":"Transgenic P. falciparum overexpressing PfHK-GFP demonstrated that PfHK is cytosolic (contradicting previous reports). Overexpression of PfHK reduced toxicity of 2-deoxy-D-glucose ~2-fold. However, overexpression of PfHK did not increase the rate of phospho-[14C]2-DG accumulation, whereas overexpression of the glucose transporter PfHT did, establishing that glucose transport (PfHT), not PfHK activity, is the rate-limiting step for glucose entry into glycolysis.","method":"Transgenic parasite lines expressing PfHK-GFP or overexpressing native PfHK; [14C]2-DG phosphorylation assays; fluorescence microscopy for localization; parasite growth assays","journal":"FEBS letters","confidence":"High","confidence_rationale":"Tier 2 / Strong — transgenic parasite lines with multiple orthogonal methods (localization, metabolic flux assay, growth assay), internally controlled with PfHT comparison","pmids":["23954294"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"}]},{"year":2013,"finding":"PfHK (55.3 kDa) is inhibited by its reaction products (product inhibition). Isobenzothiazolinone compounds inhibit PfHK with IC50s <1 µM; inhibition was reversible by dilution but not by a reducing agent, indicating the mechanism does not involve covalent modification. Several of these inhibitors also inhibit P. falciparum growth in vitro.","method":"In vitro enzyme inhibition assays; reversibility tested by dilution and reducing agent treatment; parasite growth inhibition assays","journal":"Antimicrobial agents and chemotherapy","confidence":"Medium","confidence_rationale":"Tier 1–2 / Moderate — in vitro enzymatic assays with mechanistic characterization (reversibility, reducing agent test), single lab","pmids":["23716053"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2020,"finding":"Cryo-EM structures of Plasmodium vivax HK (PvHK) at ~3 Å resolution revealed a unique tetrameric organization (~220 kDa) that can exist in open or closed quaternary conformational states, distinct from human hexokinases. The active site of PvHK resembles that of mammalian counterparts despite the unique quaternary architecture.","method":"Cryo-EM structural determination at ~3 Å resolution; two conformational states resolved","journal":"IUCrJ","confidence":"High","confidence_rationale":"Tier 1 / Moderate — cryo-EM structure at near-atomic resolution, two conformational states characterized, single lab but rigorous structural method","pmids":["32431829"],"is_preprint":false,"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2023,"finding":"The first crystal structure of P. falciparum HK (PfHK) was determined, revealing a Plasmodium-specific insertion that lines the active site and is proposed to play a role in ATP binding and to affect the tetrameric interface (suggesting inter-monomer communication). S-glutathionylation and S-nitrosation (oxidative post-translational modifications) of PfHK were confirmed to have an inhibitory effect on enzymatic activity.","method":"X-ray crystallography (first 3D structure of PfHK); enzymatic activity assays after S-glutathionylation and S-nitrosation treatment; structural analysis of the Plasmodium-specific insertion","journal":"International journal of molecular sciences","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure determination combined with functional enzyme activity assays demonstrating inhibitory effect of oxPTMs, single lab with multiple orthogonal methods","pmids":["37628920"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"finding":"Iso-mukaadial acetate (IMA) binds to recombinant PfHK with a KD of ~0.397 µM as measured by microscale thermophoresis; betulinic acid (BA) showed no detectable interaction with PfHK under the same conditions. FTIR confirmed compound-induced changes in PfHK secondary structure for IMA.","method":"Recombinant PfHK expression and purification; microscale thermophoresis (MST) binding assays; FTIR spectroscopy; molecular docking","journal":"BMC chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding measurement (MST) plus secondary structure analysis (FTIR), single lab, two orthogonal methods","pmids":["39827161"],"is_preprint":false,"attribution":[{"species":"Plasmodium falciparum","strain":"NF54"}]}],"current_model":"P. falciparum HK (PfHK, PF3D7_0624000) is a cytosolic, ~55 kDa enzyme that catalyzes the first step of glycolysis (phosphorylation of glucose to glucose-6-phosphate using ATP), possesses distinct kinetic properties from the human enzyme (higher Km for glucose, ~26% amino acid identity to human HK I), assembles as a tetramer (shown for the P. vivax ortholog by cryo-EM), contains a Plasmodium-specific insertion at its active site that contributes to ATP binding and inter-monomer communication (revealed by the first PfHK crystal structure), is subject to inhibitory oxidative post-translational modifications (S-glutathionylation and S-nitrosation), is a target of cytosolic thioredoxin-1, and limits parasitic glucose flux into glycolysis less than the upstream glucose transporter PfHT does, with its activity driving GSSG reduction via the pentose shunt in normal (but not G6PD-deficient) infected erythrocytes."},"narrative":{"mechanistic_narrative":"PF3D7_0624000 (HK) encodes the P. falciparum hexokinase, a single-copy, single-exon gene on chromosome 8 that catalyzes the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, the committed first step of parasite glycolysis, and is expressed in asexual blood stages [PMID:1475005]. The parasite enzyme is kinetically distinct from the host erythrocyte hexokinase, with a substantially higher Km for glucose (431 vs 98 µM) and only ~26% amino acid identity to human hexokinase I, and its activity is markedly elevated in infected relative to uninfected erythrocytes [PMID:3316204, PMID:1475005]. Beyond feeding glycolysis, HK-derived glucose-6-phosphate supplies the pentose shunt to regenerate NADPH for glutathione (GSSG) reduction, with GSSG reduction rate proportional to HK activity in normal but not G6PD-deficient infected cells [PMID:3316204]. HK is cytosolic, and metabolic-flux comparisons in transgenic parasites establish that glucose transport by PfHT, not HK activity, is the rate-limiting step for glucose entry into glycolysis [PMID:23954294]. The enzyme is redox-regulated: it is a target of cytosolic thioredoxin-1, and S-glutathionylation and S-nitrosation inhibit its activity [PMID:20307687, PMID:37628920]. Structurally, the first PfHK crystal structure revealed a Plasmodium-specific active-site insertion implicated in ATP binding and inter-monomer communication [PMID:37628920], and cryo-EM of the P. vivax ortholog showed a distinctive tetrameric architecture adopting open and closed states [PMID:32431829]. These species-specific kinetic, structural, and regulatory features underpin sustained interest in HK as an antiplasmodial drug target, validated by inhibition with glucose analogs, isobenzothiazolinones, and iso-mukaadial acetate binding [PMID:18713952, PMID:23716053, PMID:39827161].","teleology":[{"year":1957,"claim":"Establishing that infected erythrocytes contain elevated hexokinase activity addressed whether the parasite contributes its own glucose-phosphorylating capacity to explain increased glucose consumption.","evidence":"Enzyme inhibition assays on P. berghei-infected rodent hemolysates compared with yeast hexokinase","pmids":["13413145"],"confidence":"Medium","gaps":["Did not distinguish parasite from host enzyme molecularly","Rodent-model finding, not P. falciparum"],"attribution":[{"species":"Plasmodium berghei","strain":""}]},{"year":1987,"claim":"Demonstrating distinct kinetics and coupling to GSSG reduction answered whether the parasite enzyme is biochemically separable from the host and what downstream role its product serves.","evidence":"Enzyme kinetics on parasite lysates plus GSSG reduction assays, including G6PD-deficient comparison, in P. falciparum","pmids":["3316204","3058230"],"confidence":"Medium","gaps":["Performed on lysates, not purified enzyme","Did not define the gene or protein sequence"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":1992,"claim":"Cloning the single-copy gene and confirming it by antibody inhibition established the molecular identity of the parasite-encoded hexokinase and its divergence from the human enzyme.","evidence":"cDNA/genomic cloning, sequence analysis, Western blotting, and antibody inhibition of enzyme activity in P. falciparum","pmids":["1475005"],"confidence":"High","gaps":["No structural or catalytic-residue detail","Localization not definitively resolved"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2010,"claim":"Capturing HK as a thioredoxin-1 target raised the possibility that the glycolytic enzyme is subject to redox regulation.","evidence":"Trx-affinity chromatography with a C33S cysteine trap and LC-MS identification in P. falciparum","pmids":["20307687"],"confidence":"Low","gaps":["Single pulldown without HK-specific functional follow-up","Physiological redox effect not demonstrated in this study"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2013,"claim":"Transgenic overexpression experiments resolved HK's localization and its position in the glycolytic hierarchy, showing transport rather than phosphorylation limits glucose entry.","evidence":"PfHK-GFP and native overexpression lines with fluorescence localization, [14C]2-DG flux assays, and PfHT comparison in P. falciparum 3D7","pmids":["23954294","23716053"],"confidence":"High","gaps":["Does not address regulation of HK under nutrient stress","Inhibitor mechanism of action on the enzyme not structurally defined"],"attribution":[{"species":"Plasmodium falciparum","strain":"3D7"}]},{"year":2020,"claim":"Cryo-EM of the P. vivax ortholog answered the quaternary organization question, revealing a tetramer with open/closed states unlike human hexokinases.","evidence":"~3 Å cryo-EM structures resolving two conformational states of P. vivax HK","pmids":["32431829"],"confidence":"High","gaps":["Structure is of the vivax ortholog, not PF3D7_0624000","Functional consequence of the conformational states not defined"],"attribution":[{"species":"Plasmodium vivax","strain":""}]},{"year":2023,"claim":"The first PfHK crystal structure plus oxidative-modification assays connected a Plasmodium-specific active-site insertion to ATP binding/inter-monomer communication and confirmed inhibitory redox PTMs.","evidence":"X-ray crystallography with enzyme activity assays after S-glutathionylation and S-nitrosation in P. falciparum","pmids":["37628920"],"confidence":"High","gaps":["In vivo relevance of the oxPTMs during infection not established","Insertion's role in ATP binding inferred structurally, not mutationally tested"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]},{"year":2025,"claim":"Direct binding measurement of a candidate inhibitor advanced HK's validation as a druggable target.","evidence":"Recombinant PfHK microscale thermophoresis, FTIR, and docking with iso-mukaadial acetate versus betulinic acid in P. falciparum NF54","pmids":["39827161"],"confidence":"Medium","gaps":["No co-crystal defining the binding site","Enzyme inhibition and cellular antiplasmodial mechanism for IMA not established here"],"attribution":[{"species":"Plasmodium falciparum","strain":"NF54"}]},{"year":null,"claim":"Whether HK is genetically essential in P. falciparum and how its redox regulation operates dynamically during intraerythrocytic development remain unresolved.","evidence":"","pmids":[],"confidence":"Low","gaps":["No conditional knockout/knockdown phenotype in the corpus","Physiological triggers and reversibility of S-glutathionylation/S-nitrosation in vivo undefined","No PfHK inhibitor co-structure to guide selective drug design"],"attribution":[{"species":"Plasmodium falciparum","strain":""}]}],"mechanism_profile":{"molecular_activity":[],"localization":[],"pathway":[],"complexes":["Homotetramer (open/closed states shown for the P. vivax ortholog by cryo-EM) [#12]"],"partners":["CYTOSOLIC THIOREDOXIN-1 (PFTRX-1) — CAPTURED AS HK-BINDING REDOX TARGET [#9]"],"other_free_text":["Single-copy, single-exon gene on chromosome 8; ~26% amino acid identity to human hexokinase I [#2]","Kinetically distinct from host erythrocyte HK: higher Km for glucose (431 µM) [#0]","Activity feeds pentose shunt for NADPH-dependent GSSG reduction in normal but not G6PD-deficient infected cells [#0]","Not the rate-limiting step for glucose entry into glycolysis; PfHT transport is [#10]","Inhibited by S-glutathionylation and S-nitrosation; harbors a Plasmodium-specific active-site insertion implicated in ATP binding and inter-monomer communication [#13]","Subject to product inhibition; inhibited in vitro by glucose analogs (2-DG, 2-FG), isobenzothiazolinones, and iso-mukaadial acetate [#8, #11, #14]"]}},"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":["hexokinase"],"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 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PfHK activity is ~25-fold higher in infected erythrocytes than in control uninfected erythrocytes. The rate of GSSG reduction in infected erythrocyte lysates was proportional to hexokinase activity, establishing that PfHK-driven glucose-6-phosphate production feeds the pentose shunt to generate NADPH for glutathione reduction.\",\n      \"method\": \"Enzyme kinetics assays on parasite lysates; GSSG reduction assays correlated with parasitemia and hexokinase activity; comparison with G6PD-deficient infected red cell lysates\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzyme kinetics with functional consequence (GSSG reduction), single lab, two orthogonal approaches (kinetics + GSSG reduction assay)\",\n      \"pmids\": [\"3316204\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"Hexokinase activity is extremely elevated in P. falciparum-infected erythrocytes compared to uninfected red cells, and an electrophoretically distinct band of hexokinase activity is present, indicating parasite-encoded HK distinct from the host enzyme.\",\n      \"method\": \"Enzyme activity assays and electrophoretic analysis of glycolytic enzymes in infected vs. uninfected erythrocytes and isolated parasites\",\n      \"journal\": \"Blood\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — electrophoretic separation plus enzyme activity assays, single lab, two orthogonal methods\",\n      \"pmids\": [\"3058230\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1992,\n      \"finding\": \"PfHK is encoded by a single-copy gene on chromosome 8, translated from a single 1.5-kb exon within a 3-kb mRNA during asexual stages, and the protein has ~26% amino acid identity to human hexokinase I. Polyclonal antibodies against recombinant PfHK partially inhibited hexokinase activity in a P. falciparum lysate, confirming the cloned gene encodes the parasite hexokinase.\",\n      \"method\": \"cDNA/genomic DNA cloning, sequence analysis, Western blotting with poly- and monoclonal antibodies, antibody inhibition of enzyme activity\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1–2 / Strong — gene cloning, protein-level confirmation by Western blot, functional confirmation by antibody inhibition of enzymatic activity, multiple orthogonal methods\",\n      \"pmids\": [\"1475005\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1994,\n      \"finding\": \"PfHK expression and cellular localization were characterized during blood-stage development of P. falciparum.\",\n      \"method\": \"Not fully specified in available abstract (cellular localization study)\",\n      \"journal\": \"Molecular and biochemical parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — abstract not retrievable; only title available, insufficient detail to assess methods rigorously\",\n      \"pmids\": [\"8183319\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1957,\n      \"finding\": \"Hexokinase activity in hemolysates of P. berghei-infected mouse/rat erythrocytes is much higher than in normal erythrocytes, confirming the presence of a parasite hexokinase. Several antimalarial compounds (but not proguanil itself; its metabolite 10,580 was effective) inhibit P. berghei hexokinase in a manner similar to yeast hexokinase. The inhibition by mepacrine was reduced when ATP concentration was raised, while inhibition by compound 10,732 was independent of ATP concentration.\",\n      \"method\": \"Enzyme inhibition assays on hemolysates of P. berghei-infected rodent erythrocytes; comparison with yeast hexokinase\",\n      \"journal\": \"British journal of pharmacology and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — enzyme inhibition assays with multiple compounds and ATP competition analysis, single lab\",\n      \"pmids\": [\"13413145\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1979,\n      \"finding\": \"Parasite-specific hexokinase activity was demonstrated in P. berghei parasites isolated from infected mouse erythrocytes, providing an explanation for increased glucose consumption in infected cells.\",\n      \"method\": \"Enzyme assays on parasites isolated by hemolysin lysis; serology to confirm purity\",\n      \"journal\": \"The Journal of parasitology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single enzyme assay, single lab, no mechanistic detail beyond detection\",\n      \"pmids\": [\"376819\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1990,\n      \"finding\": \"In P. berghei, hexokinase was partially purified from isolated parasites and kinetically characterized; its kinetic properties differ significantly from erythrocyte hexokinase, and it is less efficiently inhibited. The enzyme is present in the cytosolic fraction of the isolated parasite.\",\n      \"method\": \"Partial purification, enzyme kinetics, subcellular fractionation\",\n      \"journal\": \"Biomedica biochimica acta\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — partial purification plus kinetic characterization and fractionation, single lab\",\n      \"pmids\": [\"2143651\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1997,\n      \"finding\": \"P. berghei hexokinase was purified from isolated parasites; its activity is 35-fold higher than in normal mouse erythrocytes and 5-fold higher than in P. berghei-infected erythrocytes. The enzyme has a subunit molecular weight of ~47 kDa and Km for ATP of 2 mM. Maximal activity is present in the cytosolic fraction.\",\n      \"method\": \"Purification (Sephadex G-200), SDS-PAGE, enzyme kinetics, subcellular fractionation\",\n      \"journal\": \"Acta veterinaria Hungarica\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — enzyme purification with kinetic characterization and fractionation, single lab\",\n      \"pmids\": [\"9270135\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"2-fluoro-2-deoxy-D-glucose (2-FG) and 2-deoxy-D-glucose (2-DG) inhibit glucose phosphorylation by PfHK; a trend was observed between antiplasmodial activity of glucose analogs and their ability to inhibit hexokinase-mediated glucose phosphorylation. This validates PfHK and early glycolytic steps as antiplasmodial drug targets.\",\n      \"method\": \"Hexokinase inhibition assays with glucose analogs; glucose accumulation assays in intraerythrocytic parasites; correlation with antiplasmodial activity\",\n      \"journal\": \"The Journal of pharmacology and experimental therapeutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro enzyme inhibition plus cellular glucose phosphorylation assay, single lab, two orthogonal approaches\",\n      \"pmids\": [\"18713952\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"PfHK was captured as a target protein of cytosolic thioredoxin-1 (PfTrx-1) using a cysteine mutant (C33S) Trx-affinity chromatography approach, identifying PfHK as a potential redox-regulated glycolytic enzyme.\",\n      \"method\": \"Trx-affinity chromatography with a cysteine mutant (mixed disulfide trapping), LC-MS protein identification\",\n      \"journal\": \"Parasitology international\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single affinity pulldown with MS identification, no direct functional follow-up on PfHK specifically\",\n      \"pmids\": [\"20307687\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Transgenic P. falciparum overexpressing PfHK-GFP demonstrated that PfHK is cytosolic (contradicting previous reports). Overexpression of PfHK reduced toxicity of 2-deoxy-D-glucose ~2-fold. However, overexpression of PfHK did not increase the rate of phospho-[14C]2-DG accumulation, whereas overexpression of the glucose transporter PfHT did, establishing that glucose transport (PfHT), not PfHK activity, is the rate-limiting step for glucose entry into glycolysis.\",\n      \"method\": \"Transgenic parasite lines expressing PfHK-GFP or overexpressing native PfHK; [14C]2-DG phosphorylation assays; fluorescence microscopy for localization; parasite growth assays\",\n      \"journal\": \"FEBS letters\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — transgenic parasite lines with multiple orthogonal methods (localization, metabolic flux assay, growth assay), internally controlled with PfHT comparison\",\n      \"pmids\": [\"23954294\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}]\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"PfHK (55.3 kDa) is inhibited by its reaction products (product inhibition). Isobenzothiazolinone compounds inhibit PfHK with IC50s <1 µM; inhibition was reversible by dilution but not by a reducing agent, indicating the mechanism does not involve covalent modification. Several of these inhibitors also inhibit P. falciparum growth in vitro.\",\n      \"method\": \"In vitro enzyme inhibition assays; reversibility tested by dilution and reducing agent treatment; parasite growth inhibition assays\",\n      \"journal\": \"Antimicrobial agents and chemotherapy\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1–2 / Moderate — in vitro enzymatic assays with mechanistic characterization (reversibility, reducing agent test), single lab\",\n      \"pmids\": [\"23716053\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cryo-EM structures of Plasmodium vivax HK (PvHK) at ~3 Å resolution revealed a unique tetrameric organization (~220 kDa) that can exist in open or closed quaternary conformational states, distinct from human hexokinases. The active site of PvHK resembles that of mammalian counterparts despite the unique quaternary architecture.\",\n      \"method\": \"Cryo-EM structural determination at ~3 Å resolution; two conformational states resolved\",\n      \"journal\": \"IUCrJ\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — cryo-EM structure at near-atomic resolution, two conformational states characterized, single lab but rigorous structural method\",\n      \"pmids\": [\"32431829\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"The first crystal structure of P. falciparum HK (PfHK) was determined, revealing a Plasmodium-specific insertion that lines the active site and is proposed to play a role in ATP binding and to affect the tetrameric interface (suggesting inter-monomer communication). S-glutathionylation and S-nitrosation (oxidative post-translational modifications) of PfHK were confirmed to have an inhibitory effect on enzymatic activity.\",\n      \"method\": \"X-ray crystallography (first 3D structure of PfHK); enzymatic activity assays after S-glutathionylation and S-nitrosation treatment; structural analysis of the Plasmodium-specific insertion\",\n      \"journal\": \"International journal of molecular sciences\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure determination combined with functional enzyme activity assays demonstrating inhibitory effect of oxPTMs, single lab with multiple orthogonal methods\",\n      \"pmids\": [\"37628920\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Iso-mukaadial acetate (IMA) binds to recombinant PfHK with a KD of ~0.397 µM as measured by microscale thermophoresis; betulinic acid (BA) showed no detectable interaction with PfHK under the same conditions. FTIR confirmed compound-induced changes in PfHK secondary structure for IMA.\",\n      \"method\": \"Recombinant PfHK expression and purification; microscale thermophoresis (MST) binding assays; FTIR spectroscopy; molecular docking\",\n      \"journal\": \"BMC chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding measurement (MST) plus secondary structure analysis (FTIR), single lab, two orthogonal methods\",\n      \"pmids\": [\"39827161\"],\n      \"is_preprint\": false,\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"NF54\"}]\n    }\n  ],\n  \"current_model\": \"P. falciparum HK (PfHK, PF3D7_0624000) is a cytosolic, ~55 kDa enzyme that catalyzes the first step of glycolysis (phosphorylation of glucose to glucose-6-phosphate using ATP), possesses distinct kinetic properties from the human enzyme (higher Km for glucose, ~26% amino acid identity to human HK I), assembles as a tetramer (shown for the P. vivax ortholog by cryo-EM), contains a Plasmodium-specific insertion at its active site that contributes to ATP binding and inter-monomer communication (revealed by the first PfHK crystal structure), is subject to inhibitory oxidative post-translational modifications (S-glutathionylation and S-nitrosation), is a target of cytosolic thioredoxin-1, and limits parasitic glucose flux into glycolysis less than the upstream glucose transporter PfHT does, with its activity driving GSSG reduction via the pentose shunt in normal (but not G6PD-deficient) infected erythrocytes.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"PF3D7_0624000 (HK) encodes the P. falciparum hexokinase, a single-copy, single-exon gene on chromosome 8 that catalyzes the ATP-dependent phosphorylation of glucose to glucose-6-phosphate, the committed first step of parasite glycolysis, and is expressed in asexual blood stages [#2]. The parasite enzyme is kinetically distinct from the host erythrocyte hexokinase, with a substantially higher Km for glucose (431 vs 98 µM) and only ~26% amino acid identity to human hexokinase I, and its activity is markedly elevated in infected relative to uninfected erythrocytes [#0, #2]. Beyond feeding glycolysis, HK-derived glucose-6-phosphate supplies the pentose shunt to regenerate NADPH for glutathione (GSSG) reduction, with GSSG reduction rate proportional to HK activity in normal but not G6PD-deficient infected cells [#0]. HK is cytosolic, and metabolic-flux comparisons in transgenic parasites establish that glucose transport by PfHT, not HK activity, is the rate-limiting step for glucose entry into glycolysis [#10]. The enzyme is redox-regulated: it is a target of cytosolic thioredoxin-1, and S-glutathionylation and S-nitrosation inhibit its activity [#9, #13]. Structurally, the first PfHK crystal structure revealed a Plasmodium-specific active-site insertion implicated in ATP binding and inter-monomer communication [#13], and cryo-EM of the P. vivax ortholog showed a distinctive tetrameric architecture adopting open and closed states [#12]. These species-specific kinetic, structural, and regulatory features underpin sustained interest in HK as an antiplasmodial drug target, validated by inhibition with glucose analogs, isobenzothiazolinones, and iso-mukaadial acetate binding [#8, #11, #14].\",\n  \"teleology\": [\n    {\n      \"year\": 1957,\n      \"claim\": \"Establishing that infected erythrocytes contain elevated hexokinase activity addressed whether the parasite contributes its own glucose-phosphorylating capacity to explain increased glucose consumption.\",\n      \"evidence\": \"Enzyme inhibition assays on P. berghei-infected rodent hemolysates compared with yeast hexokinase\",\n      \"pmids\": [\"13413145\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Did not distinguish parasite from host enzyme molecularly\", \"Rodent-model finding, not P. falciparum\"],\n      \"attribution\": [{\"species\": \"Plasmodium berghei\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"Demonstrating distinct kinetics and coupling to GSSG reduction answered whether the parasite enzyme is biochemically separable from the host and what downstream role its product serves.\",\n      \"evidence\": \"Enzyme kinetics on parasite lysates plus GSSG reduction assays, including G6PD-deficient comparison, in P. falciparum\",\n      \"pmids\": [\"3316204\", \"3058230\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Performed on lysates, not purified enzyme\", \"Did not define the gene or protein sequence\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 1992,\n      \"claim\": \"Cloning the single-copy gene and confirming it by antibody inhibition established the molecular identity of the parasite-encoded hexokinase and its divergence from the human enzyme.\",\n      \"evidence\": \"cDNA/genomic cloning, sequence analysis, Western blotting, and antibody inhibition of enzyme activity in P. falciparum\",\n      \"pmids\": [\"1475005\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No structural or catalytic-residue detail\", \"Localization not definitively resolved\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Capturing HK as a thioredoxin-1 target raised the possibility that the glycolytic enzyme is subject to redox regulation.\",\n      \"evidence\": \"Trx-affinity chromatography with a C33S cysteine trap and LC-MS identification in P. falciparum\",\n      \"pmids\": [\"20307687\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Single pulldown without HK-specific functional follow-up\", \"Physiological redox effect not demonstrated in this study\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Transgenic overexpression experiments resolved HK's localization and its position in the glycolytic hierarchy, showing transport rather than phosphorylation limits glucose entry.\",\n      \"evidence\": \"PfHK-GFP and native overexpression lines with fluorescence localization, [14C]2-DG flux assays, and PfHT comparison in P. falciparum 3D7\",\n      \"pmids\": [\"23954294\", \"23716053\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Does not address regulation of HK under nutrient stress\", \"Inhibitor mechanism of action on the enzyme not structurally defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"3D7\"}]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Cryo-EM of the P. vivax ortholog answered the quaternary organization question, revealing a tetramer with open/closed states unlike human hexokinases.\",\n      \"evidence\": \"~3 Å cryo-EM structures resolving two conformational states of P. vivax HK\",\n      \"pmids\": [\"32431829\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structure is of the vivax ortholog, not PF3D7_0624000\", \"Functional consequence of the conformational states not defined\"],\n      \"attribution\": [{\"species\": \"Plasmodium vivax\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"The first PfHK crystal structure plus oxidative-modification assays connected a Plasmodium-specific active-site insertion to ATP binding/inter-monomer communication and confirmed inhibitory redox PTMs.\",\n      \"evidence\": \"X-ray crystallography with enzyme activity assays after S-glutathionylation and S-nitrosation in P. falciparum\",\n      \"pmids\": [\"37628920\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of the oxPTMs during infection not established\", \"Insertion's role in ATP binding inferred structurally, not mutationally tested\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Direct binding measurement of a candidate inhibitor advanced HK's validation as a druggable target.\",\n      \"evidence\": \"Recombinant PfHK microscale thermophoresis, FTIR, and docking with iso-mukaadial acetate versus betulinic acid in P. falciparum NF54\",\n      \"pmids\": [\"39827161\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No co-crystal defining the binding site\", \"Enzyme inhibition and cellular antiplasmodial mechanism for IMA not established here\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"NF54\"}]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether HK is genetically essential in P. falciparum and how its redox regulation operates dynamically during intraerythrocytic development remain unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No conditional knockout/knockdown phenotype in the corpus\", \"Physiological triggers and reversibility of S-glutathionylation/S-nitrosation in vivo undefined\", \"No PfHK inhibitor co-structure to guide selective drug design\"],\n      \"attribution\": [{\"species\": \"Plasmodium falciparum\", \"strain\": \"\"}]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0004396\", \"supporting_discovery_ids\": [0, 2, 8, 11]},\n      {\"term_id\": \"GO:0005524\", \"supporting_discovery_ids\": [13]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [10, 6, 7]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"GO:0006096\", \"supporting_discovery_ids\": [0, 2, 10]},\n      {\"term_id\": \"GO:0006098\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"complexes\": [\n      \"Homotetramer (open/closed states shown for the P. vivax ortholog by cryo-EM) [#12]\"\n    ],\n    \"partners\": [\n      \"Cytosolic thioredoxin-1 (PfTrx-1) — captured as HK-binding redox target [#9]\"\n    ],\n    \"other_free_text\": [\n      \"Single-copy, single-exon gene on chromosome 8; ~26% amino acid identity to human hexokinase I [#2]\",\n      \"Kinetically distinct from host erythrocyte HK: higher Km for glucose (431 µM) [#0]\",\n      \"Activity feeds pentose shunt for NADPH-dependent GSSG reduction in normal but not G6PD-deficient infected cells [#0]\",\n      \"Not the rate-limiting step for glucose entry into glycolysis; PfHT transport is [#10]\",\n      \"Inhibited by S-glutathionylation and S-nitrosation; harbors a Plasmodium-specific active-site insertion implicated in ATP binding and inter-monomer communication [#13]\",\n      \"Subject to product inhibition; inhibited in vitro by glucose analogs (2-DG, 2-FG), isobenzothiazolinones, and iso-mukaadial acetate [#8, #11, #14]\"\n    ]\n  }\n}","audit_flag":null,"strains":[{"strain":"Pf3D7","gene_id":"PF3D7_0624000","name":null,"product":"hexokinase","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PbANKA","gene_id":"PBANKA_1122900","name":null,"product":"hexokinase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null},{"strain":"PkH","gene_id":"PKNH_1126200","name":null,"product":"hexokinase, putative","uniprot_acc":null,"chromosome":null,"start_pos":null}],"gene_symbol":"HK","evaluation":null}