Kinetics of Acetophenone Assay for ATAs

The kinetic parameters of ATAs were measured using the acetophenone assay and using pyruvate with concentrations from 0.001 to 0.1 mM as the amino acceptor. The reactions were performed in the same way as in the activity measurements except for using variable pyruvate concentration. Reactions (200 µL) were performed on a UV-compatible 96-well microtiter plate at 37 °C without shaking. The UV-absorbance of the assay mixtures was measured at 245 nm in 20-s intervals for 10 min, and the initial linear portion of absorbance-vs-time graphs was used to calculate the rate of acetophenone formation. The initial rates were plotted against pyruvate concentrations, and kinetic parameters were calculated using Prism 10 software.