Characterization of trehalose phosphorylase from Schizophyllum commune

During growth on D-glucose, the basidiomycete Schizophyllum commune produce s an intracellular alpha,alpha-trehalose phosphorylase. Specific phosphoryl ase activity increases steadily during the exponential growth phase, up to a maximum of approx. 0.08 unit/mg of protein, and decreases after the avail able D-glucose in the medium has been fully depleted. The variation with ti me of the concentrations of intracellular alpha,alpha-trehalose and P-1 is reciprocal to that of trehalose phosphorylase activity, indicating that the enzyme makes temporary use of the pool of alpha,alpha-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 k Da protein, which seems to lack regulation at the level of enzyme activity. The enzyme catalyses the reversible phosphorolysis of alpha,alpha-trehalos e into alpha-D-glucose 1-phosphate and alpha-D-glucose in the absence of co factors, with a catalytic-centre activity at 30 degrees C of 14 s(-1). Doub le-reciprocal analysis of the initial velocities for trehalose phosphorolys is and synthesis yields intersecting patterns, and no exchange reaction occ urs between alpha-D-glucose 1-phosphate and the phosphate analogue arsenate . Therefore trehalose phosphorylase operates by a ternary-complex, rather t han a Ping-Pong, kinetic mechanism. The specificity constants (k(cat)/K-m) of phosphate (6000 M-1.s(-1)) and alpha-D-glucose 1-phosphate (3500 M-1.s(- 1)) compared with those of alpha,alpha-trehalose (161 M-1.s(-1)) and D-gluc ose (260 M-1.s(-1)), together with the inhibition by NaCl, which is competi tive with respect to phosphate with a K-i of 67 mM, suggest an important ro le for ionic enzyme-phosphate interactions in the catalytic mechanism of tr ehalose phosphorylase. The isolated enzyme requires alpha,alpha-trehalose ( 0.1-0.3 M) for its conformational stability.