Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity

Emmanuel Oluwadare Balogun*, Daniel Ken Inaoka, Tomoo Shiba, Suzumi M. Tokuoka, Fuyuki Tokumasu, Kimitoshi Sakamoto, Yasutoshi Kido, Paul A.M. Michels, Yoh Ichi Watanabe, Shigeharu Harada, Kiyoshi Kita

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Background In general, glycerol kinases (GKs) are transferases that catalyze phospho group transfer from ATP to glycerol, and the mechanism was suggested to be random bi-bi. The reverse reaction i.e. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GKs the mechanism of Trypanosoma brucei gambiense glycerol kinase (TbgGK) was shown to be in an ordered fashion, and proceeding via autophosphorylation. From the unique reaction mechanism of TbgGK, we envisaged its potential to possess phosphatase activity in addition to being a kinase. Methods Our hypothesis was tested by spectrophotometric and LC-MS/MS analyses using paranitrophenyl phosphate (pNPP) and TbgGK's natural substrate, G3P respectively. Furthermore, protein X-ray crystallography and site-directed mutagenesis were performed to examine pNPP binding, catalytic residues, and the possible reaction mechanism. Results In addition to its widely known and expected phosphotransferase (class II) activity, TbgGK can efficiently facilitate the hydrolytic cleavage of phosphoric anhydride bonds (a class III property). This phosphatase activity followed the classical Michaelis-Menten pattern and was competitively inhibited by ADP and G3P, suggesting a common catalytic site for both activities (phosphatase and kinase). The structure of the TGK-pNPP complex, and structure-guided mutagenesis implicated T276 to be important for the catalysis. Remarkably, we captured a crystallographic molecular snapshot of the phosphorylated T276 reaction intermediate. Conclusion We conclude that TbgGK has both kinase and phosphatase activities. General significance This is the first report on a bifunctional kinase/phosphatase enzyme among members of the sugar kinase family.

Original languageEnglish
Pages (from-to)2830-2842
Number of pages13
JournalBBA - General Subjects
Issue number11
Early online date2 Aug 2017
Publication statusPublished - Nov 2017


  • Enzyme mechanism
  • Glycerol kinase
  • Parasite metabolism
  • Phosphatase
  • Trypanosoma brucei
  • X-ray crystallography


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