Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario N9B 3P4, Canada.
J Phys Chem B. 2010 Sep 2;114(34):11196-206. doi: 10.1021/jp102399h.
Glycosidic bonds are remarkably resistant to cleavage by chemical hydrolysis. Glycoside hydrolases catalyze their selective hydrolysis in oligosaccharides, polysaccharides, and glycoconjugates by following nonredox catalytic pathways or a net redox-neutral catalytic pathway using NAD(+) and divalent metal ions as cofactors. GlvA (6-phospho-alpha-glucosidase) is a glycosidase belonging to family GH4 and follows a regioselective redox-neutral mechanism of glycosidic-bond hydrolysis that favors alpha- over beta-glycosides. Its proposed catalytic mechanism can be divided into two half-reactions: the first one activates the glucopyranose ring by successively forming intermediates that are oxidized at the 3-, 2-, and 1-positions of the ring, which ultimately facilitate the heterolytic deglycosylation. The second half-reaction is essentially the reverse of the first half-reaction, beginning with the pyranose ring hydroxylation at the anomeric carbon, and it is followed by 3-reduction and regeneration of the active forms of the catalytic site and its cofactors. We investigated the NAD(+)-dependent redox mechanism of glycosidic bond hydrolysis as catalyzed by GlvA through the combined application of density functional theory and a self-consistent reaction field to a large active-site model obtained from the crystallographic structure of the enzyme, then we applied natural bond orbital and second-order perturbation analyses to monitor the electron flow and change in oxidation state on each atomic center along the reaction coordinate to rationalize the energetics and regioselectivity of this catalytic mechanism. We find that in GlvA, the redox catalytic mechanism of hydrolysis is driven by the gradual strengthening of the axial endo-anomeric component within the hexose ring along the reaction coordinate to facilitate the heterolytic dissociation of the axial C1-O bond. In addition, the combined influence of specific components of the generalized anomeric effect fully explains the regioselectivity observed in the catalytic activity of GlvA.
糖苷键对化学水解的断裂具有很强的抵抗力。糖苷水解酶通过遵循非氧化还原催化途径或使用 NAD(+)和二价金属离子作为辅助因子的净氧化还原中性催化途径，在寡糖、多糖和糖缀合物中催化它们的选择性水解。GlvA（6-磷酸-α-葡萄糖苷酶）是一种属于 GH4 家族的糖苷酶，遵循糖苷键水解的区域选择性氧化还原中性机制，有利于α-糖苷而不是β-糖苷。其提出的催化机制可分为两个半反应：第一个半反应通过依次形成氧化在环的 3-、2-和 1-位的中间体来激活葡萄糖吡喃环，最终促进异裂去糖基化。第二个半反应实质上是第一个半反应的逆反应，从吡喃糖环在端基碳原子上的羟化开始，随后是 3-还原和催化部位及其辅助因子的活性形式的再生。我们通过组合应用密度泛函理论和自洽反应场，对从酶的晶体结构获得的大型活性位点模型进行研究，研究了 GlvA 催化的糖苷键水解的 NAD(+)依赖的氧化还原机制，然后我们应用自然键轨道和二阶微扰分析来监测电子流沿反应坐标上每个原子中心的氧化态变化，以合理化该催化机制的能量学和区域选择性。我们发现，在 GlvA 中，水解的氧化还原催化机制是由六元环中环轴内端基成分沿反应坐标逐渐增强驱动的，这有利于轴 C1-O 键的异裂解离。此外，广义端基效应的特定成分的综合影响充分解释了 GlvA 催化活性中观察到的区域选择性。
