Steady-state kinetics of prenyltransferase

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)}