Figure 6.

Reductive half‐reaction of pyranose dehydrogenase from A. meleagris followed by stopped‐flow spectroscopy after mixing with sugar substrates (A) GLC, (B) GAL, (C) ARA and (D) MGP. A solution of fully oxidized enzyme (30 μm) was mixed with buffer containing various sugar concentrations (0.15, 0.3, 0.6, 1.2, 2.4 and 5 mm in 50 mm potassium phosphate buffer, pH 7.0) at 4 °C. All given concentrations are reported after mixing. The reaction was monitored at 463 nm. The kinetic traces from right to left correspond to increasing sugar concentrations. To evaluate the experimental traces (black), a double‐exponential fit was used. For all sugars, the first phase k _obs(1) was the FAD reduction with a large absorbance decrease at 463 nm, which was used to calculate k _app,4 °C. The experimental traces were simulated with berkeley madonna, version 8.3.14, according to Kinetic Model 1_red (red traces) with the parameter sets listed in Table 5. The inset shows the plot of the pseudo first‐order rate constants k _obs(1) versus the concentrations of the respective sugar.