Figure 5. The effect of substrate concentration on the rate of product formation by Rv1086_{L84A, L85F}.

Reaction mixtures contained 50 mM MOPS (pH 7.9), 5.0 mM dithiothreitol, 10 mM sodium orthovanadate, 1 mM MgCl₂, 0.3% Triton X-100, 10 μg protein and the indicated amount of GPP in a final volume of 50 μl. The reactions were incubated at 37°C for 30 min and stopped with 1ml of water saturated with NaCl, extracted with 1 ml of butanol saturated with water, and an aliquot was taken for liquid scintillation spectrometry. The K_m (inset) was calculated using nonlinear regression analysis (GraFit 5.0.13).