Barr B K, Wolfgang D E, Piens K, Claeyssens M, Wilson D B
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, USA.
Biochemistry. 1998 Jun 30;37(26):9220-9. doi: 10.1021/bi980063i.
The determination of the high-resolution structure of the Thermomonospora fusca endocellulase E2 catalytic domain makes it ideal for exploring cellulase structure-function relationships. Here we present binding parameters (Kd, DeltaH degrees, and DeltaS degrees) describing the interaction of E2 with 4-methylumbelliferyl glycosides, determined by titrating the quenching of ligand fluorescence in equilibrium binding experiments. Quenched MU(Glc)2/E2 complexes were used as indicators in displacement titrations to measure the binding of natural glycosides and also of a nonhydrolyzable cellotetraose analogue. Binding of MU(Glc)2 and cellotriose were also determined by titration calorimetry. The results show that E2 binds glycosides exclusively in its active-site cleft, with high affinity and specificity. The observed patterns of ligand hydrolysis and the results with MU(Glc)2 as a substrate indicated that ligands bound to E2 with their nonreducing ends in position -2, consistent with the position of cellobiose in the E2cd structure. Polymerase chain reaction (PCR) mutagenesis of the conserved residue Tyr 73 (in E2 binding subsite -1) to Phe and Ser produced enzymes with lower activity but higher binding affinities, indicating that the volume of the subsite -1 binding pocket is crucial for enzyme function. Similarly, MUXylGlc (with its xylosyl unit located in position -1) bound with 100-fold higher affinity than MU(Glc)2. These results are similar to those for the related Trichoderma reesei exocellulase CBH II. The binding data were compared with that previously reported for CBH II and interpreted in terms of the functional differences between endo- and exocellulases.
嗜热栖热放线菌内切葡聚糖酶E2催化结构域的高分辨率结构测定使其成为探索纤维素酶结构-功能关系的理想对象。在此，我们给出了描述E2与4-甲基伞形酮糖苷相互作用的结合参数（解离常数Kd、焓变ΔH°和熵变ΔS°），这些参数是通过在平衡结合实验中滴定配体荧光猝灭来确定的。猝灭的MU(Glc)2/E2复合物在置换滴定中用作指示剂，以测量天然糖苷以及一种不可水解的纤维四糖类似物的结合。MU(Glc)2和纤维三糖的结合也通过滴定热分析法测定。结果表明，E2仅在其活性位点裂隙中以高亲和力和特异性结合糖苷。观察到的配体水解模式以及以MU(Glc)2为底物的结果表明，配体以其非还原端位于-2位的方式与E2结合，这与纤维二糖在E2cd结构中的位置一致。将保守残基Tyr 73（在E2结合亚位点-1中）通过聚合酶链反应（PCR）突变为Phe和Ser，产生了活性较低但结合亲和力较高的酶，这表明亚位点-1结合口袋的体积对酶的功能至关重要。同样，MUXylGlc（其木糖基单元位于-1位）的结合亲和力比MU(Glc)2高100倍。这些结果与相关的里氏木霉外切葡聚糖酶CBH II的结果相似。将这些结合数据与先前报道的CBH II的数据进行了比较，并根据内切和外切葡聚糖酶之间的功能差异进行了解释。
