PURIFICATION AND CHARACTERIZATION OF TREHALOSE PHOSPHORYLASE FROM MICROCOCCUS VARIANS

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 beta-glucose 1-phosphate was 1:1:1:1 (molar ratio), The enzyme had high specificity for trehalose, glucose, and beta-glucose 1-phosphate. The K(m)s for trehalose, Pi, glucose, and beta-glucose 1-phosphate were 10, 3.1, 23, and 38mM, respectively, The k(cat)s were 200 s(-1) for trehalose phosphorolysis and 650 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.