Glycerol kinase of African trypanosomes possesses an intrinsic phosphatase activity

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

来源：Biochimica et Biophysica Acta-General Subjects, 2017, 1861(11): 2830-2842.

DOI：10.1016/j.bbagen.2017.07.028

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

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 Le. phospho transfer from glycerol 3-phosphate (G3P) to ADP is only physiologically feasible by the African trypanosome GK. In contrast to other GILs 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.

• 出版日期2017-11