Enzchek pyrophosphate assay kit

Assays were performed in triplicate in 50 µL of 50 mM Tris pH 7.5 containing 2 mM 15, 5 mM nitrite, 4 mM MgCl₂, and 20 µM Tri17. The tested concentrations for ATP were: 10 µM, 50 µM, 100 µM, 500 µM, and 1 mM. The inorganic pyrophosphate released from these enzymatic assays was measured continuously using the EnzChek Pyrophosphate Assay Kit (ThermoFisher). The 20X buffer, MESG substrate, purine nucleoside phosphorylase, and inorganic pyrophosphatase were added following the manufacturer protocols. Reactions were initiated with addition of nitrite and monitored at 360 nm using a SpectraMax M2 plate reader. Initial velocities were calculated using the standard curve for inorganic pyrophosphate. Kinetic parameters were determined and plotted using GraphPad Prism.

The EnzChek Pyrophosphate Assay Kit (Molecular Probes) was used to determine the steady-state kinetic parameters for prenyltransferase activity as previously described.^{27 (link)} Briefly, reaction mixtures contained 50 mM Tris HCl pH (7.5), 1 mM MgCl₂, 0.1 mM NaN₃, 200 μM 2-amino-6-mercapto-7-methylpurine ribonucleoside (MesGR), 0.002 U purine nucleoside phosphorylase, and 0.00006 U inorganic pyrophosphatase. For steady-state kinetics, a final concentration of 500 nM PvCPS variants and 1 mM DMAPP were held constant while IPP concentration ranged from 10–1000 μM. A parent mixture containing all reaction components except IPP was mixed and incubated at room temperature for 15 min. The reaction was initiated by the addition of the parent mixture (95 μL) to varied IPP concentrations (5 μL) for a 100-μL total reaction mixture. Generation of 2-amino-6-mercapto-7-methylpurine (MesG) was monitored at A360 on a Tecan Infinite M1000 multi-mode plate reader. Negative controls (without enzyme, without substrate) were carried out for each PvCPS variant and the largest rate from the negative control was subtracted as background from all other determined rates. Trials for each PvCPS variant were performed in triplicate. Initial rates were fit to the substrate inhibition enzyme kinetics equation in the Prism 9 program suite since IPP exhibited substrate inhibition.^{27 (link)}