Biosciences Center, National Renewable Energy Laboratory, Golden, Colorado 80401, USA.
J Biol Chem. 2011 Nov 25;286(47):41028-35. doi: 10.1074/jbc.M111.297713. Epub 2011 Sep 29.
Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions.
蛋白质在广泛的生物功能中利用芳香族残基来结合碳水化合物。糖苷水解酶在自然界中普遍存在，通常表现为具有芳香族残基排列的隧道、裂隙或口袋，用于加工碳水化合物。这些芳香族残基的突变通常会导致不溶性和可溶性底物的活性差异显著。然而，这些芳香族残基的热力学基础和分子水平作用仍然未知。在这里，我们通过将里氏木霉家族 6 纤维素酶（Cel6A）中的色氨酸突变为丙氨酸来计算相对配体结合自由能。催化部位附近芳香族残基的去除对配体结合自由能几乎没有影响，这表明活性位点上游的芳香族残基不直接参与结合，但在催化所需的吡喃葡萄糖环变形中发挥作用。然而，Cel6A 隧道入口和出口处芳香族残基的去除会显著影响结合亲和力，这表明这些残基分别在链获取和产物稳定化中发挥作用。结合亲和力差异所暗示的作用通过分子动力学和正常模式分析得到了证实。令人惊讶的是，我们的结果表明，芳香族-碳水化合物相互作用根据酶隧道中的位置而有很大差异。由于芳香族-碳水化合物相互作用存在于所有碳水化合物活性酶中，因此这些结果对于理解碳水化合物代谢和识别中的蛋白质结构-功能关系、自然界中的碳循环以及生物质利用的蛋白质工程策略具有重要意义。一般来说，这些结果表明，自然界采用了具有广泛结合亲和力的芳香族-碳水化合物相互作用来实现多种功能。
