FIGURE 4.

His₆-GFRP-mediated allosteric regulation of GTPCH by small molecules. A, the activity of 0.1 μm wt-GTPCH or Δ45-GTPCH was assayed in the presence or absence of 1 μm His₆-GFRP in 100 mm Tris-HCl and 3 mm Phe (pH 7.8) at 37 °C in reactions containing 5–500 μm GTP, using the kinetic microplate assay described under “Experimental Procedures.” Shown is a typical plot of reaction rate versus substrate concentration from one experiment. Values plotted are mean ± S.E. of triplicate measurements. B, the activity of 0.1 μm wt-GTPCH or Δ45-GTPCH was assayed in 1.5 mm DAHP or 1.5 mm DAHP and 3 mm Phe in 100 mm Tris-HCl (pH 7.8) at 37 °C, 1 mm GTP, in reactions containing 0.01 to 1 μm His₆-GFRP, using the kinetic microplate assay described under “Experimental Procedures.” Values are plotted as a percentage of GTPCH activity, measured in the absence of His₆-GFRP. Shown is a typical plot of reaction rate versus His₆-GFRP concentration from a single experiment. Values are mean ± S.E. of triplicate measurements. Nonlinear regression analysis was used to fit the solid curve to the data points, whereas a dotted line simply connects all points.