Institute of Chemistry, Slovak Academy of Sciences, 845 38 Bratislava, Slovak Republic.
J Am Chem Soc. 2012 Sep 19;134(37):15563-71. doi: 10.1021/ja307040m. Epub 2012 Sep 10.
In higher eukaryotes, a variety of proteins are post-translationally modified by adding O-linked N-acetylglucosamine (GlcNAc) residue to serine or threonine residues. Misregulation of O-GlcNAcylation is linked to a wide variety of diseases, such as diabetes, cancer, and neurodegenerative diseases, including Alzheimer's disease. GlcNAc transfer is catalyzed by an inverting glycosyltransferase O-GlcNAc transferase (uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylaminyltransferase, OGT) that belongs to the GT-B superfamily. The catalytic mechanism of this metal-independent glycosyltransferase is of primary importance and is investigated here using QM(DFT)/MM methods. The structural model of the reaction site used in this paper is based on the crystal structures of OGT. The entire enzyme-substrate system was partitioned into two different subsystems: the QM subsystem containing 198 atoms, and the MM region containing 11,326 atoms. The catalytic mechanism was monitored by means of three two-dimensional potential energy maps calculated as a function of three predefined reaction coordinates at different levels of theory. These potential energy surfaces revealed the existence of a concerted S(N)2-like mechanism, in which a nucleophilic attack by O(Ser), facilitated by proton transfer to the catalytic base, and the dissociation of the leaving group occur almost simultaneously. The transition state for the proposed reaction mechanism at the MPW1K level was located at C1-O(Ser) = 1.92 Å and C1-O1 = 3.11 Å. The activation energy for this passage was estimated to be ~20 kcal mol(-1). These calculations also identified, for the first time for glycosyltransferases, the substrate-assisted mechanism in which the N-acetamino group of the donor participates in the catalytic mechanism.
在高等真核生物中，各种蛋白质通过将 O-连接的 N-乙酰葡萄糖胺（GlcNAc）残基添加到丝氨酸或苏氨酸残基上来进行翻译后修饰。O-GlcNAc 修饰的失调与多种疾病有关，例如糖尿病、癌症和神经退行性疾病，包括阿尔茨海默病。GlcNAc 的转移由反转糖苷基转移酶 O-GlcNAc 转移酶（尿苷二磷酸-N-乙酰葡萄糖胺：多肽 β-N-乙酰氨基葡萄糖基转移酶，OGT）催化，OGT 属于 GT-B 超家族。这种非依赖金属的糖苷基转移酶的催化机制非常重要，本文使用 QM（DFT）/MM 方法对此进行了研究。本文中使用的反应位点结构模型基于 OGT 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 198 个原子，MM 区域包含 11326 个原子。通过在不同理论水平上计算三个预定义反应坐标的三个二维势能图来监测催化机制。这些势能表面揭示了存在协同 S（N）2 样机制，其中 O（Ser）的亲核攻击，通过质子转移到催化碱而得到促进，并且离去基团的解离几乎同时发生。在 MPW1K 水平上，所提出的反应机制的过渡态位于 C1-O（Ser）= 1.92 Å 和 C1-O1 = 3.11 Å。该过渡态的活化能估计约为 20 kcal mol(-1)。这些计算还首次确定了糖基转移酶中的底物辅助机制，其中供体的 N-乙酰氨基参与催化机制。
