Biely P, Vrsanská M, Gorbacheva I V
Biochim Biophys Acta. 1983 Feb 28;743(1):155-61. doi: 10.1016/0167-4838(83)90429-6.
The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate.
利用在还原端标记了（1-³H）的1,4-β-木寡糖，对黑曲霉酸性内切-1,4-β-木聚糖酶（1,4-β-D-木聚糖木聚糖水解酶，EC 3.2.1.8）的底物结合位点进行了研究。在单分子水解条件下测定了寡糖的键断裂频率和V/Km参数，并根据菅沼等人的方法（《生物化学杂志》（东京）（1978年）84卷，293 - 316页）用于评估亚位点亲和力。发现该酶的底物结合位点由七个亚位点组成，朝向结合木庚糖还原端单元的亚位点依次编号为-IV、-III、-II、-I、I、II和III。催化基团位于亚位点-I和I之间，其亲和力尚未确定。所有其他亚位点对木糖基残基的结合亲和力均为正值。从亚位点-II和II开始，亲和力值在两个方向上类似地降低。由于催化基团周围亲和力的这种几乎对称分布，该酶优先切割寡糖中距离两个末端最远的键。因此，酸性黑曲霉β-木聚糖酶似乎是一种以内切方式随机攻击聚合底物的内切-1,4-β-木聚糖酶。以羧甲基木聚糖为底物的粘度测量结果支持了这一结论。
