Substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus.

The substrate-binding site of endo-1,4-beta-xylanase of the yeast Cryptococcus albidus was investigated using, 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Evaluation of the affinities of ten imaginary subsites by the method of Suganuma et al. [1978, J. Biochem. (Tokyo) 84, 293--316] pointed out that the substrate-binding site of the enzyme is composed of four subsites and that the catalytic groups are localized in the centre. The imaginary subsites on the left-hand side of the binding site ('non-reducing-end' side) showed little or no affinity to bind xylosyl residues. For the subsites on the right-hand side of the binding site ('reducing-end' side) negative values of affinity were obtained, which means this region of the enzyme is unfavourable for complexing with xylosyl residues. As a consequence of the asymmetric distribution of negative values of affinity around the binding site, the enzyme displays a strong preference for attacking near the reducing end of the substrate. Regardless of the length of [1-3H]xylooligosaccharides, [1-3H]xylobiose was the prevailing reaction product at an early stage of hydrolysis, and frequency distribution of bond cleavage decreased from the second glycosidic bond towards the non-reducing end. Additional information on the substrate-binding site of C. albidus beta-xylanase was obtained by evaluating the efficiency of xylose, xylobiose, methyl beta-D-xyloside and phenyl beta-D-xyloside to serve as glycosyl acceptors in the transglycosylic reactions proceeding at high concentrations of xylotriose.