Catalytic Versatility of Bacillus pumilus β-Xylosidase: Glycosyl Transfer and Hydrolysis Promoted with a- and β-D-Xylosyl Fluoride

Bacillus pumilus β-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of β-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with a- and β-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of β-D-xylopyranosyl fluoride at a high rate, V = 6.25 μmol min^-1 mg^-1at 0 °C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon a-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, ¹H NMR spectra of a digest of a-D-xylosyl fluoride showed the substrate to be specifically converted to a-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this “inverting” enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from a-D-xylosyl fluoride to form a β-D-xylosidic product, followed by hydrolysis of the latter to produce a-D-xylose. The transient intermediate product formed enzymically from a-D-xylosyl fluoride in the presence of [¹⁴C]xylose was isolated and shown by its specific radioactivity and ^]H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-β-D-xylopyranosyl-D-xylopyranose containing one [¹⁴C]xylose residue. The results are related to our earlier findings with β-amylase, glyco-amylase, glucodextranase, and trehalase, which also had appeared to be strictly limited to catalyzing the hydrolysis of glycosidic substrates with inversion but which also were found to have functionally flexible catalytic groups capable of catalyzing nonhydrolytic glycosylation reactions by mechanisms other than for hydrolysis.