2.3.4. SAT-Coupled Continuous Assay

The SAT-coupled continuous assays were performed in buffer H with the addition of acetyl-CoA to 170 µM, DTNB to 0.5 mM, and 430 mU of HiSAT. L-OPS was used at different concentrations ranging from 10 to 500 µM, depending on the experiment. The units of HiSAT were modified in some experiments, as specified. Each reaction was set up in a microcuvette (l = 1 cm) pre-incubated in the spectrophotometer cuvette holder for 3 minutes at 37 °C before reading the baseline absorbance at 412 nm. After 3 minutes of incubation, the reactions were triggered by the addition of PSP, and the initial velocity was estimated from the linear phase of the kinetic trace. The variation in absorbance at 412 nm was used to calculate the rate of product synthesis using the extinction coefficient reported for TNB at pH 7 (14,100 M⁻¹·cm⁻¹) [35,36,37]. The specific activity of HiSAT was estimated at the same temperature and in buffer H, with the additional presence of 170 µM acetyl-CoA, 0.5 mM DTNB, and 1 mM L-serine [42,43]. A unit of HiSAT is defined as the amount of enzyme able to convert 1 µmole of acetyl-CoA in free coenzyme A in one minute in the presence of 1 mM L-Ser to give OAS at 37 °C, pH 7. The detection range of the assay is 4–70 μM phosphate.