Infinite m1000 multimode plate reader

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)}

The enzymatic activity of active recombinant caspase-9 (Enzo Life Sciences) was evaluated by the Caspase-Glo® 9 Assay kit from Promega, in which catalysis of a substrate by caspase-9 releases a substrate for luciferase (aminoluciferin), resulting in the luciferase reaction and a detectable luminescence emission in vitro. Ten microliters of serial dilutions of designed protein in caspase assay buffer [50 mM of Hepes, 100 mM of NaCl, 1 mM of EDTA, 1 mM DTT with 0.1% of CHAPS and 10% of glycerol (pH 7.4)] were mixed with 2.5 μL of active caspase-9 solution in caspase assay buffer. This mixture was incubated at room temperature for 15 min. Luminogenic Z-LEHD substrate was added with 1:1 ratio to give final caspase-9 concentration of 2.5 unit/reaction (according to the manufacturer’s instructions). This mixture was incubated at 295 K for 1 h without light, and luminescence from substrate cleavage was then determined by a Tecan Infinite M-1000 multimode plate reader.