Purification and Characterization of Trehalose Phosphorylase from Micrococcus varians

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Trehalose phosphorylase (EC 2. 4. 1. 64), which catalyzes the reversible reaction of phosphorolysis and synthesis of trehalose, was purified to homogeneity from a cell-free extract of Micrococcus varians strain No. 39. The enzyme was shown to have a molecular weight of 570, 000 to 580, 000 by gel filtration, and to have a subunit of molecular weight of 105, 000 by SDS-polyacrylamide gel electrophoresis. The stoichiometry of the reaction between trehalose, Pi, glucose, and β-glucose 1-phosphate was 1 : 1 : 1 : 1 (molar ratio). The enzyme had high specificity for trehalose, glucose, and β-glucose 1-phosphate. The Kms for trehalose, Pi, glucose, and β-glucose 1-phosphate were 10, 3. 1, 23, and 38mM, respectively. The Kcats were 200s-1 for trehalose phosphorolysis and 660 s - 1 for trehalose synthesis. The enzyme was inhibited by validamycin A, validoxylamine A, 1-deoxynojirimycin, and Cu2+ during trehalose phosphorolysis, and by Cu2+, Zn2+, and Ni2+ during trehalose synthesis. Inhibition competitive against trehalose was noted with validamycin A, validoxylamide A, and 1-deoxynojirimycin. Initial velocity, product inhibition, and dead-end inhibition studies suggested that both trehalose phosphorolysis and trehalose synthesis proceeded through an ordered Bi Bi mechanism.
社団法人日本農芸化学会の論文
1995-10-23