FIGURE 3.

A, UV-visible absorbance spectrum of SepCysS revealing the presence of bound PLP in the overexpressed recombinant enzyme. B, imaged TLC plate showing time-dependent formation of Cys-tRNA^Cys catalyzed by SepCysS. Conditions were as follows: 50 mm Tris (pH 7.5), 20 mm KCl, 10 mm MgCl₂, 5 mm ATP, 3 mm phosphoserine, 5 mm DTT, 10 μm tRNA^Cys, and 20 μm SepRS. After 5 min, Cys-tRNA^Cys formation was initiated by the addition of SepCysS to 20 μm and sodium sulfide to 5 mm. The percentage conversion to Cys-tRNA^Cys is determined by the ratio of the intensities for Sep-AMP and Cys-AMP and converted to molarity by multiplying by the concentration of Sep-tRNA^Cys determined from the plateau aminoacylation value. C, plot of the velocity of Cys-tRNA^Cys synthesis catalyzed by SepCysS versus enzyme concentration under steady-state conditions. No activity is observed at enzyme concentrations below 50 nm. A constant concentration of 5 μm was used.