Kinetic Analysis of CB1954 Prodrug Reduction

CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader using the method that we have previously described [32 (link),33 (link),34 (link)]. Product formation was measured at 420 nm over time to determine the -Menten kinetic parameters of the CB1954 prodrug with either NfnB-Cys or YfkO-Cys. CB1954 (0.1–10 mM), NADH (400 µM) and PB (50 mM, pH 7.2) were combined and incubated at 37 °C for 3 min before purified NfnB-Cys or YfkO-Cys (10 µg/mL). Hydroxylamine yield per second was determined by calculating the change in absorbance over 20 s and the molar extinction coefficient, which is the same for both products (ε = 1200 M⁻¹ cm⁻¹ at 420 nm) [12 (link),13 (link),15 (link),25 (link),32 (link)]. Data was analysed using SigmaPlot 12 (SPSS, Systat Software Inc., Chicago, USA) where a non-linear regression tool was used to generate a Michaelis-Menten hyperbolic curve and a report containing the important kinetic information of the system under test. Coefficient of Variability (CoV) for the kinetics is 7.06%.