All chemicals were purchased from Sigma (St Louis, MO) unless otherwise stated. AOPP was from Wako (Osaka, Japan). Methodologies for the determination of KAT‐I enzyme activity, and inhibition analysis by HPLC‐UV was based on our previous report,45 and was originally described by Okuno et al. and Li and Li.47, 48 A typical reaction mixture of 30 µL containing 15 µL of substrate mixture (5 mM KYN, 2 mM α‐ketobutyrate, 40 mM PLP), 2 µg purified KAT‐1 in 5 µL solution and 10 µL of inhibitor at different concentrations was prepared in 200 mM phosphate buffer at pH 8. Each inhibitor was incorporated into the reaction mixture at final concentrations of 0.0004, 0.004, 0.037, 0.37, 3.7, 37, 111, 333, 1000, and 3000 µM.
KAT‐1 was pre‐incubated with each inhibitor (or buffer) for 10 minutes at room temperature before adding the substrate mixture. The mixture was incubated at 45°C for 10 minutes and the reaction was stopped by adding 30 µL of 1 M trichloroacetic acid. The resulting mixture was centrifuged at 15,000 rcf at 4°C for 5 min, and the supernatant used as the analyte on the HPLC. A positive control reaction (inhibitor substituted with buffer) and negative control reaction (KAT‐1 solution and inhibitor substituted with buffer) were simultaneously performed with all assays. All reactions were performed in triplicates.
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