FIGURE 4.
Activity of IDI-2 apoenzyme in the presence of FMN (white), 1-deazaFMN (grey), and 5-deazaFMN (black). The incubation mixture contained 4.9 μM apo-IDI-2, 5 mM NADPH (or 5 or 50 mM dithionite), 50 μM [1-14C]IPP, 5 mM MgCl2, and 100 μM flavin coenzyme in 0.1 M HEPES buffer (pH 7.0). All reactions were incubated for 10 min at 37 °C, quenched with 25% HCl:MeOH, saturated with NaCl, and extracted with toluene or petroleum ether. The radioactivity of a 0.3 mL sample removed from the organic extracts was measured by liquid scintillation counting (see Experimental Procedures for details).