DiFMUP assay

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.