Steady-state enzyme kinetics

Activities towards varying concentrations of para-nitrophenyl phosphate (pNPP) were measured in 100 µL assay buffer (25 mM HEPES pH 7.5, 2 mM TCEP, 1 mM EDTA, and 150 mM NaCl) at 30 °C. Enzyme concentrations were chosen to achieve absorbance values within the linear response range while satisfying the steady-state assumption where substrate consumption was < 1%. Reactions were initiated by enzyme addition and stopped with 1 N NaOH. Absorbance at 405 nm was measured on a Synergy H1 microplate reader (BioTek) and converted to product concentration using a para-nitrophenol standard curve. Initial rates were plotted as a function of substrate concentration and fit with the Michealis-Menten equation in GraphPad Prism (Version 8) to determine k_cat and K_M.

Activities towards varying concentrations of the synthetic phosphopeptide HT(pSer)PIKSIG (Genscript Corp) were measured in 50 µL assay buffer at 30 °C essentially as described above for pNPP, except substrate consumption was limited to 10% due to limited assay sensitivity and the reaction was stopped with 100 µL Biomol Green (Enzo Life Sciences). Absorbance was measured at 640 nm and converted to product with a sodium phosphate standard curve. k_cat and K_M were calculated as described above.

Activities towards varying concentrations of 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP, Thermo Fisher Scientific) were performed under the same conditions described above for pNPP with the exception of VHR, which was assayed in 50 mM Bis–Tris (pH 6.0), 1 mM DTT, and 100 mM NaCl. All enzyme concentrations were 0.5 nM, except for PsCdc14 and VHR, which were assayed at 2 and 5 nM, respectively. Fluorescence intensity was measured continuously on a Synergy H1 microplate reader (BioTek) with excitation and emission wavelengths set at 358 and 450 nm, respectively. Fluorescence intensity was converted to product concentration using a 6,8-difluoro-4-methylumbelliferone standard curve. Background fluorescence was subtracted from each reaction and initial rates were calculated from the slope of the linear portion of the product concentration versus time plots. k_cat and K_M were calculated as described above.