ITC experiments were carried out as described.38 (link) Briefly, enzyme was dialyzed extensively against 20 mM HEPES, pH 8.0, 300 mM NaCl, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine) supplemented with 500 μM MnCl2 and 10% (v/v) DMSO, and the dialysate was used to prepare fresh working solutions of various inhibitors. The manganese ion was utilized instead of iron since it would not oxidize during the time frame of these experiments. Titration was carried out at 0.42–1.1 mM compound [I] concentration and enzyme [E] concentration of 25–80 μM. Compounds were injected into protein or buffer (to measure the heat of dilution) in 25 steps of 4 μL volume using a MicroCal Auto-iTC200. Binding affinity (KD), stoichiometry (N), and binding enthalpy (ΔH) were determined by fitting the data using the ITC data analysis module of Origin version 7.0 (OriginLab Corp.).
For each ITC experiment, we performed a test run to figure out the optimal enzyme and inhibitor concentrations to be used and range of titrations. Each curve is fitted for 20 data points. Due to the small differences in the KD values of very similar chemical compounds, the data presented resulted from experiments done at the same time using the same batch of enzymes, which is essential to avoid the variation from batches of enzyme preparation.
For each ITC experiment, we performed a test run to figure out the optimal enzyme and inhibitor concentrations to be used and range of titrations. Each curve is fitted for 20 data points. Due to the small differences in the KD values of very similar chemical compounds, the data presented resulted from experiments done at the same time using the same batch of enzymes, which is essential to avoid the variation from batches of enzyme preparation.
