Abstract：
In the barley β-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures β-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric β-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-β-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.
摘要：
在大麦β-D-葡聚糖葡萄糖水解酶中，糖苷水解酶家族3（GH3）酶，Trp286/Trp434夹钳确保β-D-葡糖苷结合，这是植物生长发育过程中底物水解的基础。我们采用诱变，高分辨率X射线晶体学，和多尺度分子建模方法，以检查在GH3水解酶中操作的底物产物辅助的持续催化过程中异构β-D-葡糖苷的结合和构象行为。酶动力学表明，W434H突变体保留了广泛的特异性，而W434A表现为严格的(1,3)-β-D-葡糖苷酶。纳米级反应物运动的研究表明，持续合成能力对色氨酸钳的突变特异性改变敏感。虽然野生型和W434H利用侧腔进行葡萄糖置换和（1,3）连接的水解产物通过催化位点滑动而不解离，与它们的高水解率一致，W434A不采用持续催化。GH3水解酶的系统学分析揭示了色氨酸钳的进化优势，赋予广泛的特异性，高催化效率，和可持续发展。
