Hamre Anne Grethe, Frøberg Emil Ebbestad, Eijsink Vincent G H, Sørlie Morten
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway.
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway.
Arch Biochem Biophys. 2017 Apr 15;620:35-42. doi: 10.1016/j.abb.2017.03.011. Epub 2017 Mar 28.
Glycoside hydrolases (GHs) catalyze the hydrolysis of glycosidic bonds and are key enzymes in carbohydrate metabolism. Efficient degradation of recalcitrant polysaccharides such as chitin and cellulose is accomplished due to synergistic enzyme cocktails consisting of accessory enzymes and mixtures of GHs with different modes of action and active site topologies. The substrate binding sites of chitinases and cellulases often have surface exposed aromatic amino acids and a tunnel or cleft topology. The active site of the exo-processive chitinase B (ChiB) from Serratia marcescens is partially closed, creating a tunnel-like catalytic cleft. To gain insight in the fundamental principles of substrate binding in this enzyme, we have studied the contribution of five key residues involved in substrate binding and tunnel formation to the thermodynamics of substrate binding. Mutation of Trp, Phe, Trp and Glu, which are all part of the tunnel walls, resulted in significant less favorable conformational entropy change (ΔS°) upon binding (-TΔΔS° = ∼5 kcal/mol). This suggest that these residues are important for the structural rigidity and pre-shaping of the tunnel prior to binding. Mutation of Asp, which, by forming a hydrogen bond to Trp is crucial in the active-site tunnel roof, resulted in a more favorable ΔS° relative to the wild type (-TΔΔS° = -2.2 kcal/mol). This shows that closing the tunnel-roof comes with an entropy cost, as previously suggested based on the crystal structures of GHs with tunnel topologies in complex with their substrates.
糖苷水解酶（GHs）催化糖苷键的水解，是碳水化合物代谢中的关键酶。由于由辅助酶以及具有不同作用模式和活性位点拓扑结构的GH混合物组成的协同酶混合物，几丁质和纤维素等难降解多糖得以有效降解。几丁质酶和纤维素酶的底物结合位点通常具有表面暴露的芳香族氨基酸以及隧道或裂隙拓扑结构。粘质沙雷氏菌的外切几丁质酶B（ChiB）的活性位点部分封闭，形成了类似隧道的催化裂隙。为了深入了解该酶中底物结合的基本原理，我们研究了参与底物结合和隧道形成的五个关键残基对底物结合热力学的贡献。色氨酸、苯丙氨酸、色氨酸和谷氨酸均为隧道壁的一部分，它们的突变导致结合时构象熵变（ΔS°）明显不利（-TΔΔS° = ∼5 kcal/mol）。这表明这些残基对于结合前隧道的结构刚性和预成型很重要。天冬氨酸通过与色氨酸形成氢键，在活性位点隧道顶部起关键作用，其突变导致相对于野生型更有利的ΔS°（-TΔΔS° = -2.2 kcal/mol）。这表明封闭隧道顶部伴随着熵的代价，正如之前基于具有隧道拓扑结构的GH与其底物复合物的晶体结构所提出的那样。
