Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states.
Role of active site residues in the glycosylase step of T4 endonuclease V. Computer simulation studies on ionization states.
复制标题
活性位点残基在 T4 核酸内切酶 V 糖基化步骤中的作用。电离态的计算机模拟研究。
作者：
Fuxreiter,M;Warshel,A;Osman,R
T4内切酶V(EndoV)是一种碱基切除修复酶，能从损伤的DNA中去除胸腺嘧啶二聚体(TD)。为了阐明活性中心残基在催化中的作用，用半显微版的蛋白质偶极子−朗之万偶极子方法(PDLD/S)计算了它们的pKa。详细分析了电荷−电荷相互作用、构象重排、蛋白质弛豫和DNA结合等不同效应对pK的贡献。活性中心残基的荷电在复合体中不如在游离酶中有利。N端的pKaf由游离酶的8.01降至复合体的6.52，而Glu-23的pKaof由1.52升至7.82，说明关键残基在糖基化酶步骤的反应态是中性的。这些PKA与反应的最佳pH范围一致，并支持N-末端作为亲核剂。质子化形式的Glu-23与5‘Td的糖的O4’形成氢键，从而起到增加C1‘的正电荷，从而加速亲核取代的作用。此外，中性的Glu-23很可能是质子化O4‘以诱导完成EndoV糖基化酶步骤所需的环开放的候选者。带正电荷的Arg-22和Arg-26为离开碱基提供了有利的静电环境。为了区分SN1和SN2糖基酶步骤的机制，计算了5‘Td的质子化O2的能量。该酶对中性胸腺嘧啶的稳定作用为∼3.6kcal/m ol，而对质子化胸腺嘧啶的稳定作用为∼6.6kcal/m ol。因此，质子化胸腺嘧啶中间体的形成是不可能的，这表明糖基化酶步骤的SN2反应机制。
T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pKa's were evaluated using the semimicroscopic version of the protein dipoles−Langevin dipoles method (PDLD/S). Contributions of different effects to the pKasuch as charge−charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pKaof the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pKaof Glu-23 increased from 1.52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pKa's are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4‘ of the sugar of 5‘ TD and can play a role in increasing the positive charge of C1‘ and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4‘ to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between SN1 and SN2 mechanisms of the glycosylase step the energetics of protonating O2 of 5‘ TD was calculated. The enzyme was found to stabilize the neutral thymine by ∼3.6 kcal/mol, whereas it destabilizes the protonated thymine by ∼6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an SN2 reaction mechanism for the glycosylase step.