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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry.
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Mapping glycoside hydrolase substrate subsites by isothermal titration calorimetry.

机译：通过等温滴定热分析法绘制糖苷水解酶底物亚位点。

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摘要

Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis. The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (DeltaH(a)) and allows the determination of the binding constant (free energy), entropy, and stoichiometry. In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6. The change in the heat capacity of binding (DeltaC(p) = DeltaH/DeltaT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures. Because hydrophobic stacking interactions are associated with negative DeltaC(p), the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme. The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis.
机译：将热力学参数与结构和生化数据相关联，可以更好地了解底物结合及其对催化的贡献。由于涉及多种相互作用和作用力，因此分析碳水化合物与蛋白质或酶的结合是一项特殊的挑战。等温滴定热量法（ITC）可直接测量结合焓（DeltaH（a）），并可以确定结合常数（自由能），熵和化学计量。在这项研究中，我们使用ITC阐明了从嗜热脂肪芽孢杆菌T-6中分离的10个家族的两个木聚糖酶的木糖结合热力学。通过在不同温度下使用ITC测量来确定一个糖单位不同的木糖的结合热容量的变化（DeltaC（p）= DeltaH / DeltaT）。由于疏水堆积相互作用与负DeltaC（p）相关，因此数据使我们能够基于酶的晶体结构预测结合亚位点的底物结合偏好。所提出的位置结合偏好与突变体在不同的亚位点缺少芳香结合残基相一致，并且色氨酸荧光分析也支持了该突变体。

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