Determination of kinetic parameters for maltotriose and higher malto-oligosaccharides in the reactions catalyzed by α-D-glucan phosphorylase from potato

Article abstract of DOI:10.1016/0008-6215(91)84131-W

For kinetic studies on its synthetic and phosphorolytic reactions, α-D-glucan phosphorylase from potatoes was purified chromatographically until free of D-enzyme. Purified maltotriose (G₃) is a poor primer in the phosphorylase-catalyzed synthetic reaction, showing an anomalous time course and making previous attempts to determine its kinetic parameters unsuccessful. In the present work the true rate of the G₃-primed reaction was obtained from linear plots obtained by incorporating a sufficient quantity of β-amylase in the digest to eliminate the more rapidly reacting G₄ formed from the G₃. A K(m) value of 9.4 ± 0.8 mM for G₃ was calculated from the data by a nonlinear least-squares method. Kinetic parameters for a series of higher malto-oligosaccharides (G₄-G₈) were also determined in both the synthetic and the phosphorolytic directions. A large change in the values of K(m) and V/e was seen on going from G₃ to G₄ for the synthetic reaction, and from G₄ to G₅ for the phosphorolytic. For the higher saccharides the V/e values do not vary strongly with increasing d.p., while the K(m) values tend to decrease, as has seen in the reactions of other plant phosphorylases. For kinetic studies on its synthetic and phosphorolytic reactions α-D-glucan phosphorylase from potatoes was purified chromatographically until free of D-enzyme. Purified maltotriose (G₃) is a poor primer in the phosphorylase-catalyzed synthetic reaction, showing an anomalous time course and making previous attempts to determine its kinetic parameters unsuccessful. In the present work the true rate of the G₃-primed reaction was obtained from linear plots obtained by incorporating a sufficient quantity of β-amylase in the digest to eliminate the more rapidly reacting G₄ formed from the G₄ A K_m value of 9.4 ± 0.8 mM for G₃ was calculated from the data by a nonlinear least-squares method. Kinetic parameters for a series of higher malto-oligosaccharides (G₄-G₃) were also determined in both the synthetic and the phosphorolytic directions. A large change in the values of K_m and V/e was seen on going from G₃ to G₄ for the synthetic reaction, and from G₄ to G₃ for the phosphorolytic. For the higher saccharides the V/e values do not vary strongly with increasing d.p.. while the K_m values tend to decrease, as has seen in the reactions of other plant phosphorylases.