Kinetic Analysis of HsUMPS Enzyme

The enzyme kinetics of HsUMPS enzyme were monitored by absorbance changes over time at 279 nm for the production of UMP and at 295 nm for the consumption of orotate. These reactions were conducted at 25 °C on a Synergy Neo2 microplate reader (BioTek) or SpectraMax M2 (Molecular Devices). The standard assay conditions contained 50 mM Tris pH 8.0, 100 mM NaCl, 2 mM MgCl₂. For kinetic assays, the mixture consisted of either 100 μM PrPP with varying concentrations of orotate (0–200 μM) or varying concentrations of OMP (0–200 μM). In all cases, the reaction was initiated by the addition of protein (5–50 pmol). Absorbance data were collected in triplicate, averaged, and plotted as mean ± standard error. To calculate kinetic parameters, initial rate date (before 10% reaction completion) was fit with the Michaelis–Menten equation.