FIGURE 6.

Phenylalanine binding and activation of PheH variants. A, effect of the concentration of phenylalanine on the weight-average sedimentation coefficient (s_w) of wild type (▵), L62V (○), E44Q (●), A47G (▴), H64N (□), and L48V (■) RDPheH(25–117). The lines are from fits of the data to s_w = s_m + Δs_w × [Phe]/(K_Phe + [Phe]). The value of s_m was set to 1.3 based on the s_w values in the absence of phenylalanine. For H64N and L48V RDPheH(25–117), the s_w value of the dimer was assumed to be identical to that for wild-type RDPheH(25–117). The data for wild-type RDPheH(25–117) are from Ref. 25. B, representative fluorescence changes upon binding of phenylalanine to wild-type PheH (▵), A47G PheH (▴), H64N PheH (□), and L48V PheH (■) in 0.2 m HEPES, pH 7.5, at 25 °C. The lines are from fits of the data to Δfluorescence = ΔFl_max x× [Phe]ⁿ/(K_actⁿ + [Phe]ⁿ). C, activation of PheH variants by phenylalanine. Each enzyme (25 μm) was incubated with 250 μm phenylalanine at 23 °C for 10 min before being diluted 100-fold into assay mix containing 1 mm phenylalanine and all other assay components, and the initial rate of tyrosine formation was determined.