The substrat-binding site of endo-1,4-β-xylanase of the yeast Cryptococcus albidus was investigated using, 1,4-β-xylooligosaccharides (1-3H)- labeled 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 xylosylresidues. For the subsites on the right-hand side of the binding site (‘reducing end’ side) negaive 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 β-xylanase was obtained by evaluating the efficiency of xylose, xylobiose, methyl β-d-xyloside and phenyl β-d-xyloside to serve as glycosyl acceptors in the transglycosylic reactions proceeding at high concentrations of xylotriose.