Purification and Characterization of Trehalose Phosphorylase from<i>Micrococcus varians</i>

Abstract

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 660s−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.