Zonisamide (min. 98% purity by HPLC, Lot # KWM4684) was purchased from Wako Chemicals (Richmond, VA) and used without further purification. All experiments with zonisamide were performed using freshly prepared aqueous solutions as recommended from previous studies5 (link). Other chemicals used in this study were purchased from Sigma-Aldrich. Purified preparations of MAO used in this study were recombinant enzymes expressed in Pichia pastoris as described for human MAO B16 , human MAO A17 , rat MAO A18 , rat MAO B29, and zebrafish MAO11 . Enzyme assays were performed at 25 °C in 50 mM phosphate, 0.5% (w/v) reduced Triton X-100 at air saturation.
Enzyme assays were carried out spectrophotometrically using kynuramine as substrate (Δε316 = 11,800 M−1-cm−1) for MAO A and for zebrafish MAO whereas benzylamine was used as the substrate (Δε250 = 12,800 M−1-cm−1) for MAO B. Inhibition experiments were performed with at least five concentrations of zonisamide. All kinetic and inhibition data were analyzed using non-linear least squares regression fits to the Michaelis-Menten equation using GraphPad Prism 5.0 software. The inhibition data exhibited, statistically, the best fit to a competitive inhibition mechanism with r2 values of 0.96–0.99.
Human MAO B crystals were grown as previously reported14 (link) in the presence of saturating concentrations of zonisamide and data collection performed at the ESRF and SLS synchrotron facilities. The structure was determined by molecular replacement as previously described14 (link). Statistical values for the structural determination (1.8 Å resolution) are provided inTable 2 .
Enzyme assays were carried out spectrophotometrically using kynuramine as substrate (Δε316 = 11,800 M−1-cm−1) for MAO A and for zebrafish MAO whereas benzylamine was used as the substrate (Δε250 = 12,800 M−1-cm−1) for MAO B. Inhibition experiments were performed with at least five concentrations of zonisamide. All kinetic and inhibition data were analyzed using non-linear least squares regression fits to the Michaelis-Menten equation using GraphPad Prism 5.0 software. The inhibition data exhibited, statistically, the best fit to a competitive inhibition mechanism with r2 values of 0.96–0.99.
Human MAO B crystals were grown as previously reported14 (link) in the presence of saturating concentrations of zonisamide and data collection performed at the ESRF and SLS synchrotron facilities. The structure was determined by molecular replacement as previously described14 (link). Statistical values for the structural determination (1.8 Å resolution) are provided in
