FIG. 6.
A coupled assay measured the UDP-GlcNAc 2-epimerase activity of MMP0705. (A) Reaction progress curves show activities for coupled epimerase and dehydrogenase reactions containing 3 mM UDP-GlcNAc (filled circles) and 1.5 mM UDP-GlcNAc (open circles). Control reactions without MMP0705 enzyme (filled squares) or without UDP-GlcNAc (open squares) show background levels of activity. The reaction mixtures contained 3 to 5 mM NAD+, 0.4 μg MMP0705-His6, and 150 μg His6-MMP0706 with buffer salts, as described in the text. The reduction of NAD+ to NADH was monitored by following the increase in absorbance at 340 nm. (B) Hill plot of the rate of MMP0705-catalyzed epimerization of UDP-GlcNAc. Initial rate data were fitted to the Hill equation by nonlinear regression. The Hill coefficient was 2.7, the KM for UDP-GlcNAc was 0.36 mM, and the kcat was 3.4 s−1.