Research Article
Complexed Crystal Structure of Saccharomyces cerevisiae Dihydroorotase with Inhibitor 5-Fluoroorotate Reveals a New Binding Mode
Figure 5
Fluorescence titration of ScDHOase with 5-FOA. (a) The fluorescence emission spectra of ScDHOase with 5-FOA of different concentrations (0–200 μM; 0, 10, 20, 50, 75, 100, 125, 150, 175, and 200 μM). The decrease in intrinsic fluorescence of protein was measured at 324 nm upon excitation at 280 nm with a spectrofluorimeter. The fluorescence intensity emission spectra of ScDHOase significantly quenched with 5-FOA. (b) The fluorescence emission spectra of ScDHOase-R18A with 5-FOA of different concentrations (0–200 μM). (c) The fluorescence emission spectra of ScDHOase-T106A with 5-FOA of different concentrations (0–200 μM). ScDHOase-R18A and ScDHOase-T106A individually displayed strong intrinsic fluorescence with a peak wavelength of 324.5 and 328 nm when excited at 280 nm. (d) An aliquot amount of 5-FOA was added to the enzyme solution for determining Kd. Kd was obtained by the following equation: ΔF = ΔFmax − Kd (ΔF/[5-FOA]). Data points are an average of 2-3 determinations within 10% error.
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