Figure 2
Activation of valine and methionine by WT-LeuRS.
(a) Estimate of valine activation parameters –kcat and KM. The separate kinetic parameters could not be accurately determined due to high KM value and low valine solubility. (b) A second order rate constant kcat/KM was determined to assess the catalytic efficiency of methionine and valine activation. The average value of three independent experiments for each data point is shown and the error bar represents S.E.M. (c) Isoleucine misincorporation was monitored using Leu-3 SILAC. WT or D345A-LeuRS JW5807-2 strains were grown in the presence of 0.3 mM Leu-3 to enable complete labeling of the proteome. In the early stationary phase (marked with an arrow), isoleucine (100 mM final concentration) was added to both cultures and the samples were taken for LC-MS/MS analysis prior to the addition of isoleucine (denoted as 0 h) and 0.5, 1, 2, 3, 5 and 13 h after addition of isoleucine. (d) % of isoleucine incorporation in WT or D345A-LeuRS JW5807-2 strains.
