The ATP hydrolysis assay of TpCorC and its mutant was performed by the malachite green method (61). Briefly, malachite green dye solution was freshly prepared on the day of the experiment by mixing 0.045% malachite green, 4.2% ammonium molybdate, and 1% Triton X-100 at a volume ratio of 36:12:1. Then, 1 μM TpCorC or 1 nM apyrase (Sigma-Aldrich, USA) was prepared in buffer H [20 mM Hepes (pH 7.5), 150 mM NaCl, and 0.05% DDM]. ATP was dissolved in buffer H containing 4 mM MgCl2 at a final concentration of 4 mM. To initiate the reaction, the ATP solution was added to the protein solution at an equal volume to obtain a final reaction mixture consisting of 20 mM Hepes (pH 7.5), 2 mM MgCl2, 150 mM NaCl, 0.05% DDM, and 2 mM ATP. The samples were then incubated at room temperature, and aliquots were taken at multiple time points (5, 30, 60, 90, and 120 min). Fifty microliters of each aliquot taken at each time point was mixed with 850 μl of the malachite green dye solution and 100 μl of 34% citric acid, and the absorbance was measured at 660 nm. The absorbance standard curve for inorganic phosphate was established with standard H3PO4 solutions.

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