Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovak Republic.
Chemistry. 2013 Jun 17;19(25):8153-62. doi: 10.1002/chem.201300383. Epub 2013 Apr 24.
β1,6-GlcNAc-transferase (C2GnT) is an important controlling factor of biological functions for many glycoproteins and its activity has been found to be altered in breast, colon, and lung cancer cells, in leukemia cells, in the lymhomonocytes of multiple sclerosis patients, leukocytes from diabetes patients, and in conditions causing an immune deficiency. The result of the action of C2GnT is the core 2 structure that is essential for the further elongation of the carbohydrate chains of O-glycans. The catalytic mechanism of this metal-ion-independent glycosyltransferase is of paramount importance and is investigated here by using quantum mechanical (QM) (density functional theory (DFT))/molecular modeling (MM) methods with different levels of theory. The structural model of the reaction site used in this report is based on the crystal structures of C2GnT. The entire enzyme-substrate system was subdivided into two different subsystems: the QM subsystem containing 206 atoms and the MM region containing 5914 atoms. Three predefined reaction coordinates were employed to investigate the catalytic mechanism. The calculated potential energy surfaces discovered the existence of a concerted SN 2-like mechanism. In this mechanism, a nucleophilic attack by O6 facilitated by proton transfer to the catalytic base and the separation of the leaving group all occur almost simultaneously. The transition state for the proposed reaction mechanism at the M06-2X/6-31G** (with diffuse functions on the O1', O5', OGlu , and O6 atoms) level was located at C1-O6=1.74 Å and C1-O1=2.86 Å. The activation energy for this mechanism was estimated to be between 20 and 29 kcal mol⁻¹, depending on the method used. These calculations also identified a low-barrier hydrogen bond between the nucleophile O6H and the catalytic base Glu320, and a hydrogen bond between the N-acetamino group and the glycosidic oxygen of the donor in the TS. It is proposed that these interactions contribute to a stabilization of TS and participate in the catalytic mechanism.
β1,6-N-乙酰氨基葡萄糖转移酶（C2GnT）是许多糖蛋白生物功能的重要调控因子，其活性已在乳腺癌、结肠癌和肺癌细胞、白血病细胞、多发性硬化症患者的淋巴细胞、糖尿病患者的白细胞以及引起免疫缺陷的情况下发生改变。C2GnT 作用的结果是核心 2 结构，这对于 O-聚糖碳水化合物链的进一步延长是必不可少的。这种金属离子非依赖性糖基转移酶的催化机制至关重要，本研究采用不同理论水平的量子力学（QM）（密度泛函理论（DFT））/分子建模（MM）方法进行了研究。本报告中使用的反应位点结构模型基于 C2GnT 的晶体结构。整个酶-底物系统分为两个不同的子系统：QM 子系统包含 206 个原子，MM 区域包含 5914 个原子。使用三个预定义的反应坐标来研究催化机制。计算出的势能面发现存在协同 SN2 样机制。在这种机制中，通过向催化碱质子转移促进 O6 的亲核攻击以及离去基团的分离几乎同时发生。在所提出的反应机制的 M06-2X/6-31G**（在 O1'、O5'、OGlu 和 O6 原子上具有弥散函数）水平下，过渡态位于 C1-O6=1.74 Å 和 C1-O1=2.86 Å。该机制的活化能估计在 20 到 29 kcal mol⁻¹ 之间，具体取决于所使用的方法。这些计算还确定了亲核试剂 O6H 与催化碱 Glu320 之间的低势垒氢键以及在 TS 中供体的糖苷氧与 N-乙酰氨基之间的氢键。据提议，这些相互作用有助于 TS 的稳定并参与催化机制。
