Kinetic analysis of Kynureninase enzymes

A range of substrate concentration from 0 to 1 mM and enzyme (0.01 to 1 μM) were used to determine Michaelis-Menten kinetic parameters for the kynureninase enzymes against both kynurenine (Kyn, Sigma) and 3-Hydroxy-DL-kynurenine (DL-3-OH-Kyn Sigma). Reactions were initiated by adding 80 μl of substrate solution to 20 μL of enzyme solution in Dulbelcco’s phosphate buffered saline pH = 7.4 (DPBS). Enzymatic activity was evaluated by monitoring substrate absorbance over time using a BioTek Synergy HT 96-well plate spectrometer. Absorbance at 365 nm was monitored for Kyn degradation, and at 373 nm for DL-3-OH-Kyn. Initial rates were determined from the linear region of the reaction progress curves with <10% of substrate conversion. Michaelis-Menten parameters, k_cat and K_M, were determined using KaleidaGraph (Synergy software).