Binding of 4-methylumbelliferyl-beta-D-ribopyranoside to beta-D-xylosidase from Bacillus pumilus.

The determination of the binding parameters of 4-methylumbelliferyl-beta-D-ribopyranoside, a fluorescent ligand of beta-D-xylosidase (exo-1,4-beta-xylosidase, EC 3.2.1.37) from Bacillus pumilus, is described. A single binding site per polypeptide chain (60 000 daltons) was found and the homogeneity of the binding sites in the dimeric or tetrameric forms of the enzyme were shown. The association constants, as a function of temperature and ionic strength, were obtained from equilibrium binding experiments and compared to the kinetically determined inhibition constants. The apparent discrepancies are attributed to a temperature, ionic strength and concentration dependent shift in the dimer-tetramer equilibrium of the enzyme and different affinities of the ligand for both oligomeric forms. Sedimentation velocity experiments seem to corroborate this hypothesis.