Cheng Qianyi, DeYonker Nathan John
Department of Chemistry, University of Memphis, Memphis, TN, United States.
Front Chem. 2022 Mar 24;10:854318. doi: 10.3389/fchem.2022.854318. eCollection 2022.
Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme.
糖苷水解酶对于水解多糖中的β-1,4糖苷键以解构碳水化合物很重要。使用野生型蛋白及其E217Q突变体，通过残基相互作用网络残基选择器（RINRUS）软件构建的不同大小的量子力学簇模型，探索了糖苷水解酶家族7（GH7）的两步保留反应机制。第一步是糖基化，其中酸性残基217将一个质子供体给糖苷氧导致键断裂。在随后的去糖基化步骤中，一个水分子迁移到活性位点并攻击异头碳。基于残基相互作用的量子力学簇模型为所提出的糖苷水解酶机制带来了可靠的结构和能量结果。在最大的量子力学簇模型中，野生型蛋白及其E217Q突变体糖基化的活化自由能预计分别为19.5和31.4千卡/摩尔，这与酸性残基Glu217突变为Gln会减慢反应的实验趋势一致；并且其自由能高于去糖基化过渡态（野生型蛋白及其突变体分别为13.8和25.5千卡/摩尔）。对于突变蛋白，糖基化产生了低能量产物。这个热力学阱可能对应于在X射线晶体结构中分离出的中间状态。因此，糖基化被证实是野生型和突变型酶中的限速步骤。
