Figure 2.

Exploring PGI-like activity of rhGFAT2.A, reaction scheme of complete GFAT reaction. B–C, time course of rhGFAT2-his reaction in presence of Gln and Fru-6P (both at 3 mM) in 50 mM deuterated phosphate buffer pH 7.4 with 1 mM DTT at 25 °C, monitored by ¹H NMR spectroscopy. The times at which spectral data were acquired refer to the addition of rhGFAT2-his (100 μg) as t = 0 h. B, region of ¹H NMR spectra detailing Gln and Glu peaks. C, region of ¹H NMR spectra detailing Fru-6P, Glc-6P, and GlcN-6P peaks. D, isomerization of Fru-6P in Glc-6P catalyzed by rhGFAT2-his or rhGFAT2 without (w/o) HisTag assessed either in the absence (black bars) or in presence (gray bars) of Gln. E, ammonia release from Gln, catalyzed by rhGFAT2-his (black circles) and rhGFAT2 without (w/o) HisTag (black squares). The enzyme was incubated with increasing amounts of Gln until 10 mM, and with fixed Gln at 10 mM and variable concentrations of Fru-6P as indicated.