Figure 5.

Characteristics of the chromophoric reaction intermediate. A, UV-visible absorbance spectra of freshly isolated PDX1.1, PDX1.2, and PDX1.3, respectively, in 50 mm Tris-Cl, pH 8.0. B, Reconstitution of the observed chromophoric adduct employing free PDX1.1 (20 μm), PDX2 (20 μm), Gln (10 mm), and R5P (0.2 mm) in Tris-Cl, pH 8.0. The pentose sugar was added at time 0, and the spectra were acquired after 1.5, 4.5, 6.0, 7.5, 9.0, 12.0, 25.5, and 27 min. C, Formation of the chromophoric adduct in the presence (solid line) and absence (dashed line) of Gln (10 mm) employing PDX1.1 (10 μm), PDX2 (10 μm), and R5P (0.3 mm) in Tris buffer, pH 8.0. D, Demonstration of the catalytic competence of the chromophoric adduct; after reconstitution of the latter as described in B (taken as time 0), dl-G3P (0.3 mm) was added, and spectra were acquired at 3, 6, 9, 12, 15, 18, 19.5, and 21 min. The direction of the arrows indicates either the decrease or increase in absorbance observed.