During growth on D-glucose, the basidiomycete Schizophyllum commune produces an intracellular α,α-trehalose phosphorylase. Specific phosphorylase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the available D-glucose in the medium has been fully depleted. The variation with time of the concentrations of intracellular α,α-trehalose and Pi is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of α,α-trehalose (approx. 0.42 mmol/g dry cell) via phosphorolysis. The enzyme has been purified, 150-fold, to homogeneity in 55% yield and characterized. It is a monomeric 61 kDa protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of α,α-trehalose into α-D-glucose 1-phosphate and α-D-glucose in the absence of cofactors, with a catalytic-centre activity at 30 °C of 14 s-1. Double-reciprocal analysis of the initial velocities for trehalose phosphorolysis and synthesis yields intersecting patterns, and no exchange reaction occurs between α-D-glucose 1-phosphate and the phosphate analogue arsenate. Therefore trehalose phosphorylase operates by a ternary-complex, rather than a Ping-Pong, kinetic mechanism. The specificity constants (kcat/Km) of phosphate (6000 M-1˙s-1) and α-D-glucose 1-phosphate (3500 M-1˙s-1) compared with those of α,α-trehalose (161 M-1˙s-1) and D-glucose (260 M-1˙s-1), together with the inhibition by NaCl, which is competitive with respect to phosphate with a Ki of 67 mM, suggest an important role for ionic enzyme-phosphate interactions in the catalytic mechanism of trehalose phosphorylase. The isolated enzyme requires α,α-trehalose (0.1-0.3 M) for its conformational stability.

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