內含蛋白媒介之蛋白質接合反應中氮-硫酰基重排反應機構理論及其圓二色光譜研究

Abstract

內含蛋白(intein) 為一具備自身催化反應的蛋白質區塊，在蛋白質轉譯後修飾時的蛋白質剪接(protein splicing)中扮演關鍵的角色。內含蛋白之作用為：催化自身與外含蛋白(extein)的切割、並促成兩段外含蛋白的重組，經過剪接以形成具功能之蛋白。內含蛋白的反應過程大致可分為四大步驟：N-S acyl shift、transesterification、succinimide formation 以及 S-N rearrangement。在本研究中，我們藉由量子化學為基礎的理論計算來模擬第一步驟 N-S acyl shift 的化學反應、決定其反應中間產物與過渡態的結構、並推得反應機構。首先以蛋白質資料庫中內含蛋白的X光繞射解析晶體結構、以及液態核磁共振解析結構作為基礎，建立由蛋白質殘基及支鏈所構成的實驗模型；並藉由密度泛函理論(density functional theory, DFT)及 Møller–Plesset 二階微擾理論(the second order Møller–Plesset perturbation theory, MP2)做分子結構最佳化並建立反應位能面、同時以高階耦合叢集理論(CCSD)做單點計算。計算發現 N-S acyl shift 是為二步驟反應，其中包含 oxo-thiazolidine 中間態的生成及此中間態的開環裂解。我們也發現此中間態的生成會與一分子內的氫離子轉移同時發生，而此中間態的開環則會伴隨著一分子間的氫離子轉移。此反應在MP2的計算下所得之第一及第二活化能分別為30.8 kcal/mol以及14.9 kcal/mol；此反應的總反應熱(ΔH)為7.1 kcal/mol。

Intein is a self-catalyzed protein segment, acting as one of the key complexes in the protein splicing process and mediating both of the cleavage and the ligation of protein sequences. The process begins with an N-S acyl shift followed by transesterification, forming a branched intermediate which leads to the formation of succinimide and the S-N rearrangement. Although this four-step reaction has been previously studied, the detailed mechanisms of the first step (N-S acyl shift) and the last step (S-N acyl shift) are still unconfirmed. We report the computational study and the synchrotron radiation circular dichroism (SRCD) analysis of the N-S acyl shift in protein splicing. The models were built based on the X-ray crystallographic and solution NMR structures. The geometries of each reaction stage were located using density functional theory and second order Møller–Plesset perturbation theory. The SRCD signal reveals that the intein maintains the identical structures in different salinity. The computational results show that the N-S acyl shift is a two-step reaction, which goes through an oxothiazolidine forming step followed by a ring-opening step. The formation of the oxothiazolidine intermediate is accompanied by an intramolecular proton transfer, whereas the opening of the heterocyclic ring occurs simultaneously with an intermolecular double-proton transfer. The first and the second activation energies calculated at the level of MP2 are 30.8 kcal/mol and 14.9 kcal/mol, respectively. The overall reaction energy is 7.1 kcal/mol.