Shigeo Fujimoto

Analysis of the characteristic action of D-enzyme from sweet potato in terms of subsite theory

Abstract Disproportionating enzyme (D-enzyme, EC 2.4.1.25) was purified chromatographically from a β-amylase-deficient variety of sweet potatoes, and obtained free of amylases and glucosidases. Some heavy metal ions, particularly Hg2+ and Ag+, inactivated the enzyme. The Km value for maltrotriose was found to be 7.3m m . Analysis by h.p.l.c. of digests of malto-oligosaccharides (G3–G7) showed that, as with other D-enzymes, maltose is not formed in any case. The products from all substrates except G4 are those resulting from maltosyl transfer as the predominant reaction, wherein Gn−2 and Gn+2 are produced from Gn. The various malto-oligosaccharides undergo reaction at almost the same rates. The frequency of cleavage of the various bonds in malto-oligosaccharides was estimated from the observed molar ratios of products smaller than the substrates. A possible subsite structure for the D-enzyme is proposed on the basis of computer modelling of data describing the cleavage pattern etc.

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

Abstract 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 (G 3 ) 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 G 3 -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 G 4 formed from the G 3 . A K m value of 9.4 ± 0.8 m m for G 3 was calculated from the data by a nonlinear least-squares method. Kinetic parameters for a series of higher malto-oligosaccharides (G 4 –G 8 ) 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 G 3 to G 4 for the synthetic reaction, and from G 4 to G 5 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.