FIGURE 3.

(A) Stoichiometry of NADPH consumption versus DMAPP formation over a 30-min incubation period with IDI-2. The UV-vis assays (0.178 mL) contained 5.3 μM reconstituted IDI-2, 0.125 mM NADPH, 5 mM MgCl₂, and varying concentrations of IPP (0.05, 0.50 and 1.20 mM) in 0.1 M HEPES buffer (pH 7.0). Each assay was incubated for 30 min at 37 °C as described in the Experimental Procedures. NADPH concentration (●) was determined based on the absorbance at 340 nm. DMAPP formation was determined by the radioassay using variable amounts of [1-¹⁴C]IPP (0.05 (◇), 0.50 (□), or 1.20 (▲) mM). (B) Linear relationship between NADPH consumption and enzyme concentration during turnover. Each assay contained 0.125 mM NADPH, 1.2 mM IPP, and 5 mM MgCl₂, and variable amounts of reconstituted IDI-2 (2.0, 4.1, 9.3, 14, or 22 μM) in 0.1 M HEPES buffer (pH 7.0). NADPH consumption over a 30 min period was calculated from the total change in absorbance at 340 nm.