HK

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).

1. 1957 Medium

   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

   PMID:13413145

   Open questions at the time

   • Did not distinguish parasite from host enzyme molecularly
   • Rodent-model finding, not P. falciparum
2. 1987 Medium

   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

   PMID:3058230 PMID:3316204

   Open questions at the time

   • Performed on lysates, not purified enzyme
   • Did not define the gene or protein sequence
3. 1992 High

   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

   PMID:1475005

   Open questions at the time

   • No structural or catalytic-residue detail
   • Localization not definitively resolved
4. 2010 Low

   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

   PMID:20307687

   Open questions at the time

   • Single pulldown without HK-specific functional follow-up
   • Physiological redox effect not demonstrated in this study
5. 2013 High

   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

   PMID:23716053 PMID:23954294

   Open questions at the time

   • Does not address regulation of HK under nutrient stress
   • Inhibitor mechanism of action on the enzyme not structurally defined
6. 2020 High

   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

   PMID:32431829

   Open questions at the time

   • Structure is of the vivax ortholog, not PF3D7_0624000
   • Functional consequence of the conformational states not defined
7. 2023 High

   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

   PMID:37628920

   Open questions at the time

   • In vivo relevance of the oxPTMs during infection not established
   • Insertion's role in ATP binding inferred structurally, not mutationally tested
8. 2025 Medium

   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

   PMID:39827161

   Open questions at the time

   • No co-crystal defining the binding site
   • Enzyme inhibition and cellular antiplasmodial mechanism for IMA not established here

• Whether HK is genetically essential in P. falciparum and how its redox regulation operates dynamically during intraerythrocytic development remain unresolved.
• 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