Claeyssens M, Van Tilbeurgh H, Tomme P, Wood T M, McRae S I
Laboratorium voor Bichemie, Rijksuniversiteit Gent, Belgium.
Biochem J. 1989 Aug 1;261(3):819-25. doi: 10.1042/bj2610819.
Reaction patterns for the hydrolysis of chromophoric glycosides from cello-oligosaccharides and lactose by the cellobiohydrolases (CBH I and CBH II) purified from Trichoderma reesei and Penicillium pinophilum were determined. They coincide with those found for the parent unsubstituted sugars. CBH I enzyme from both organisms attacks these substrates in a random manner. Turnover numbers are, however, low and do not increase appreciably as a function of the degree of polymerization of the substrates. The active-site topology of the CBH I from T. reesei was further probed by equilibrium binding experiments with cellobiose, cellotriose, lactose and some of their derivatives. These point to a single interaction site (ABC), spatially restricted as deduced from the apparent independency of the thermodynamic parameters. It appears that the putative subsite A can accommodate a galactopyranosyl or glucopyranosyl group, and subsite B a glucopyranosyl group, whereas in subsite C either a glucopyranosyl or a chromophoric group can be bound, scission occurring between subsites B and C. The apparent kinetic parameters (turnover numbers) for the hydrolysis of cello-oligosaccharides (and their derivatives) by the CBH II type enzyme increase as a function of chain length, indicative of an extended binding site (A-F). Its architecture allows for specific binding of beta-(1----4)-glucopyranosyl groups in subsites A, B and C. Binding of a chromophore in subsite C produces a non-hydrolysable complex. The thermodynamic interaction parameters of some ligands common to both type of enzyme were compared: these substantiate the conclusions reached above.
测定了从里氏木霉和嗜松青霉中纯化得到的纤维二糖水解酶（CBH I和CBH II）对纤维寡糖和乳糖中发色糖苷的水解反应模式。它们与未取代的母体糖的反应模式一致。来自这两种生物体的CBH I酶以随机方式攻击这些底物。然而，周转数较低，并且不会随着底物聚合度的增加而显著增加。通过与纤维二糖、纤维三糖、乳糖及其一些衍生物的平衡结合实验，进一步探究了里氏木霉CBH I的活性位点拓扑结构。这些实验表明存在一个单一的相互作用位点（ABC），根据热力学参数的明显独立性推断，该位点在空间上受到限制。似乎假定的亚位点A可以容纳一个吡喃半乳糖基或吡喃葡萄糖基，亚位点B可以容纳一个吡喃葡萄糖基，而在亚位点C中，要么可以结合一个吡喃葡萄糖基，要么可以结合一个发色基团，断裂发生在亚位点B和C之间。CBH II型酶水解纤维寡糖（及其衍生物）的表观动力学参数（周转数）随着链长的增加而增加，这表明存在一个扩展的结合位点（A-F）。其结构允许在亚位点A、B和C中特异性结合β-(1→4)-吡喃葡萄糖基。在亚位点C中结合一个发色团会产生一个不可水解的复合物。比较了两种酶共有的一些配体的热力学相互作用参数：这些参数证实了上述结论。
