NMR experiments were conducted at a
1H frequency of 500 MHz using a Bruker Avance III HD spectrometer equipped with a BBFO probe. Experiments were conducted at 300 K.
Standard 5 mm NMR tubes (Wilmad) using a sample volume of 500 μL were used in all experiments. The pulse tip-angle calibration using the single-pulse nutation method (Bruker
pulsecal routine) was undertaken for each sample.60 (
link) All measurements were performed in triplicate.
Time course experiments were monitored by standard Bruker
1H experiments with water suppression by excitation sculpting. Unless otherwise stated, the number of transients was 16, and the relaxation delay was 2 seconds. The lag time between addition of enzyme and the end of the first experiment was usually 4 minutes. Initial rates were calculated by linear fitting using Excel 2013 (Microsoft) for data points up to 20% turnover. Kinetic parameters were obtained using the Hanes plot. Linear fitting was done using Excel 2013 (Microsoft). All NMR samples contained 190 nM ICL1, 1 mM
dl-isocitrate and 5 mM MgCl
2 buffered with 50 mM Tris-D
11 (pH 7.5) in 90% H
2O and 10% D
2O. For kinetic parameter measurements, the isocitrate concentrations ranged from 50 μM to 750 μM. For single concentration inhibition assay, 100 μM inhibitors was used. For IC
50 measurements, varying concentrations of inhibitors were used. IC
50 values were obtained by SigmaPlot 13 and fitted with the
Sigmoid, 3 parameter model.
Bhusal R.P., Patel K., Kwai B.X., Swartjes A., Bashiri G., Reynisson J., Sperry J, & Leung I.K. (2017). Development of NMR and thermal shift assays for the evaluation of Mycobacterium tuberculosis isocitrate lyase inhibitors †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7md00456g. MedChemComm, 8(11), 2155-2163.