Figure 5.

Standard enzyme assay with PSY-GGPS fusion protein. A, Time-course experiment of phytoene and GGPP formation and IPP consumption under standard enzyme assay conditions with PSY-GGPS11 fusion protein (PYGG) and 20 µm DMAPP/20 µm IPP. Round open circles represent the sum of GGPP and phytoene. Product concentrations are expressed in IPP equivalents in order to facilitate direct comparison. Note that phytoene synthesis continues with almost unchanged velocity for about 2 h. B, Substrate dependancy of recombinant PYGG. IPP was provided with 5, 10, 20, 40, 60, 80, 100, and 150 µm, incubation time was 10 min. Data (R² = 0.98) were fitted with the Michaelis-Menten equation using the GraphPad Prism software (for equations, see Methods). Results in A and B are means of three replicate experiments. C, Coomassie-stained SDS-PAGE of recombinant purified proteins of His₆-GGPS11 (35.7 kD), His₆-PSY (44.2 kD), and the fusion protein His₆-PYGG (79.6 kD).