Characterisation of hexokinase in Toxoplasma gondii tachyzoites

Tomoya Saito, Takuya Maeda, Miki Nakazawa, Tsutomu Takeuchi, Tomoyoshi Nozaki, Takashi Asai

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

We have cloned the hexokinase [E.C. 2.7.1.1] gene of Toxoplasma gondii tachyzoite and obtained an active recombinant enzyme with a calculated molecular mass of 51,465Da and an isoelectric point of 5.82. Southern blot analysis indicated that the hexokinase gene existed as a single copy in the tachyzoites of T. gondii. The sequence of T. gondii hexokinase exhibited the highest identity (44%) to that of Plasmodium falciparum hexokinase and lower identity of less than 35% to those of hexokinases from other organisms. The specific activity of the homogeneously purified recombinant enzyme was 4.04μmol/mg protein/min at 37°C under optimal conditions. The enzyme could use glucose, fructose, and mannose as substrates, though it preferred glucose. Adenosine triphosphate was exclusively the most effective phosphorus donor, and pyrophosphate did not serve as a substrate. Km values for glucose and adenosine triphosphate were 8.0±0.8μM and 1.05±0.25mM, respectively. No allosteric effect of substrates was observed, and the products, glucose 6-phosphate and adenosine diphosphate, had no inhibitory effect on T. gondii hexokinase activity. Other phosphorylated hexoses, fructose 6-phosphate, trehalose 6-phosphate which is an inhibitor of yeast hexokinase, and pyrophosphate, also did not affect T. gondii hexokinase activity. Native hexokinase activity was recovered in both the cytosol and membrane fractions of the whole lysate of T. gondii tachyzoites. This result suggests that T. gondii hexokinase weakly associates with the membrane or particulate fraction of the tachyzoite cell.

Original languageEnglish
Pages (from-to)961-967
Number of pages7
JournalInternational Journal for Parasitology
Volume32
Issue number8
DOIs
Publication statusPublished - 2002 Jun 29

Keywords

  • Apicomplexa
  • Glycolysis
  • Hexokinase
  • Protozoa
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

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