【作者】 陈喜；

【导师】 湛昌国；

【作者基本信息】 华中师范大学 ， 理论物理， 2004， 博士

【摘要】 本文运用理论化学方法研究了细胞内二级信使—腺苷3′，5′-磷酸单酯(cAMP)及相关磷酸二酯的非酶水解和酶催化水解。全文由三个章节组成：(1)cAMP非酶水解的一般反应途径；(2)溶液中环状磷酸二酯和非环状磷酸二酯碱性水解活化自由能的理论计算；(3)cAMP酶催化水解的理论研究。 第一章中我们运用第一原理电子结构计算方法研究了cAMP及相关的磷酸二酯—三甲基磷酸酯(TMP)的竞争水解反应途径，并预测了各水解途径相应的反应自由能能垒。反应坐标计算表明存在三条基本水解反应途径：(A)氢氧根离子进攻磷酸酯阴离子的磷原子(属于S_N2反应机理，反应过程中无五配位磷酸酯中间体生成)；(B)水分子直接进攻磷酸酯阴离子的磷原子(属于三步历程)；(C)水分子直接进攻磷酸酯中性分子的磷原子(属于两步历程)。气相反应计算结果表明水解途径(A)所对应的自由能能垒最高，水解途径(C)的决速步骤所对应的反应自由能能垒最低。然而，在水溶液中水解途径(A)所对应的自由能能垒变成最低，(A)和(B)为两条主要的水解反应途径。与此同时，我们还研究了磷酸酯的pK_a值及溶液反应体系的pH值对总水解速率中不同水解途径的相对贡献的影响。在中性水溶液中，cAMP的水解主要通过反应途径(A)来进行。然而，随着溶液pH值的减小，反应途径(A)对总水解速率的贡献也将减少。计算结果表明，当pH＞～3.7时，反应途径(A)的贡献最大。当pH＜～3.7，时，反应途径(B)的贡献将比反应途径(A)来得更大。计算得到的TMP在溶液中的水解自由能能垒与现有的实验值符合的非常好，这充分说明了我们的理论预测的可靠性。 第二章中主要应用量子化学从头计算方法研究了四种磷酸二酯—二甲基磷酸酯(DMP)，乙撑磷酸酯(EP)，TMP阴离子及cAMP的模型化合物(cAMPm)的碱性水解反应途径，并估算了各个反应的能垒和自由能能垒。反应坐标计算表明所有这些磷酸酯的碱性水解均遵循一步历程双分子反应机理，磷酸酯的水解由氢氧根对酯上的磷原子的亲核进攻开始。气相计算结果表明，DMP，EP和TMP的碱性水解反应的能垒高度非常接近，EP分子中环张力的影响表现不明显。随后，我们用五种不同的自洽反应场(SCRF)方法计算了这四种酯在水溶液中的碱性水解反应能垒和自由能能垒，并将计算结果与现有的实验数据进行比较。计算表明最近发展的一种表面极化和体极化SCRF方法(SVPE)的计算结果对溶质电荷等密度面数值的选择不很敏感(溶质的等电荷密度面是SVPE方法中确定溶质孔穴的大小和形状溶质电荷的唯一参数)。SVPE方法有时又被称为连续极化介质模型(FPCM)，它能够同时精确地计算表面极化和体极化作用。用FPCM方法计算的结果加上非静电作用校正得到的DMP、TMP、EP和cAMPm水解反应的活化自由能分别为32.6，31.6，24.8，29.4 kcal／mol，与现有的实验估测值～32，～32，～21～24 kcal／mol(分别对应DMP、TMP和EP的水解反应)符合得非常好。FPCM计算的结果表明溶剂化作用将极大的改变磷酸二酯碱性水解的活化自由能。前人研究DMP和TMP水解速度存在巨大差别的原因是馨博士学位论文DOCTORA工DISSERTATION溶剂化效应，而不是环内张力的影响。我们的计算结果与之一致。本工作中忽略了体极化作用影响的方法系统地过高估算了反应活化自由能，但计算所得的活化自由能的相对大小关系仍与FPCM计算值和实验结果定性地一致。其他三种使用了某种电荷重归一化的SCRF方法的计算结果也过高估算了反应活化自由能，计算所得的活化自由能的相对大小关系与FPCM计算值和实验结果明显的不同。 第三章主要应用分子对接和第一原理的电子结构方法研究cAMP的酶模型催化水解。为了获得较好的底物一受体反应前络合物的初始构型，我们使用对。AMP专一的磷酸二醋酶PDE4BZB活性区和底物分子cAMP的晶体结构(扭tP://w ww.resb.o厂g/pd的，然后分别用AutoDock和FlexX程序将cAMP分子对接到PDE酶的活性中心。两种程序的计算结果一致认为底物与酶模型结合的最佳方式是cAMP分子的磷酞氧占据活性中心镁离子的六配位八面体结构中的一个锥顶位置。为了进一步用量子化学从头算方法研究底物与受体的作用机理，酶晶体活性区的结构被简化为包含两个二价金属阳离子(znZ坏口Mg2+离子)的配位化合物模型结构，。AMP则被简化为TMP。我们对此模型结构进行了多种级别的构型优化计算，结果表明不同计算级别下优化得到的结构参数相互一致，且与晶体中的相应参数保持一致。随后，在HF/3一2 IG*级别上研究了酶模型与磷酸二酷的中性分子和阴离子的作用机理。初步计算结果表明TMP阴离子的酶催化水解是由11个反应步骤组成的历程，决速步骤为桥氢氧根离子对底物磷原子的亲核进攻。然而，此步的反应能垒高达36 kcaUmol，甚至高于cAMP碱性水解的能垒(21kcaUmof)。用cAMP代替过渡态结构中的TMP得到了相似的过渡态结构，反应能垒为39kcaFmol。对此步反应过渡态结构进行分析，我们认为造成高水解能垒的因素不是配体与受体之间的静电斥力，而是亲核反应过程中的质子转移。随后，我们研究了质子化的TMP分子(TMPH)与酶模型作用的亲核历程，结果表明在PDE酶中氢氧根离子进攻TMPH磷中心亲核反应的能垒仅?更多还原

【Abstract】 In this work, a series of theoretical methods were employed to investigate the none-enzymatic and enzymatic hydrolysis of intracellular second messenger adenosine 3’, 5’-cyclic monophosphate (cAMP) and related phosphodiesters. The thesis consists of three chapters, including (1) theoretical study of none-enzymatic hydrolysis of cAMP, (2) theoretical determination of activation free energies for alkaline hydrolysis of cyclic and acyclic phosphodiesters in aqueous solution, and (3) theoretical study of the enzymatic hydrolysis of cAMP and related phosphodiester.For the studies described in the first chapter, .we have performed a series of first-principles electronic structure calculations to study competing reaction pathways and the corresponding free energy barriers for ester hydrolysis of cAMP and related phosphodiesters including trimethylene phosphate (TMP). Reaction coordinate calculations show three fundamental reaction pathways for the ester hydrolysis, including (A) attack of a hydroxide ion at the P atom of the phosphate anion (an SN2 process without a pentacoordinated phosphorus intermediate), (B) direct attack of a water molecule at the P atom of the anion (a three-step process), and (C) direct attack of a water molecule at the P atom of the neutral ester molecule (a two-step process). The calculated energetic results show that for the reactions in the gas phase, the free energy barrier for pathway (A) is the highest and the barrier for the rate-controlling step of pathway (C) is the lowest. However, for the reactions in aqueous solution, the free energy barrier calculated for pathway (A) becomes the lowest and the two main hydrolysis pathways are (A) and (B). We also have demonstrated how pK, of the ester and pH of the reaction solution affect the relative contributions of different hydrolysis pathways to the total hydrolysis rate. Reaction pathway (A) should be dominant for the cAMP hydrolysis in neutral aqueous solution. However, the relative contribution of pathway (A) to the total hydrolysis rate should decrease with decreasing pH of the solution. When pH < ~ 3.7, the contribution of pathway (B) is larger. When PH >~ 3.7, the contribution of pathway (A) is larger. The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free energy barrier with available experimental data for hydrolysis of TMP in solution.In the second chapater, first-principles electronic structure calculations were performed to examine the reaction pathway and corresponding activation free energies for alkaline hydrolysis of representative phosphodiesters, including dimethyl phosphate (DMP), trimethylene phosphate (TMP), ethylene phosphate (EP), and a simplified model (cAMPm) of adenosine 3’, 5’-phosphate (cAMP). Reaction .coordinate calculations show that for all of these phosphodiesters the alkaline hydrolysis follows a one-step bimolecular mechanism initialized by the attack of hydroxide ion at thephosphorous atom of the ester. Five self-consistent reaction field (SCRF) methods were used to calculate the activation free energies and the calculated results were compared with available experimental data. It has been shown that the results calculated by using a recently developed SCRF method, known as the surface and volume polarization for electrostatics (SVPE) or fully polarizable continuum model (FPCM), which accurately determines both surface and volume polarization, are rather insensitive to the used solute charge isodensity contour value which determines the solute cavity size. The SVPE calculations plus nonelectrostatic interaction corrections led to activation free energies 32.6, 31.6, and 24.8 kcal/mol for DMP, TMP, and EP, respectively. The calculated activation free energies are all in good agreement with available experimentally estimated activation free energies -32, -32, and -21-24 kcal/mol for DMP, TMP, and EP, respectively. The FPCM results show that the solvation dramatically decreases the activation free energies for the alkaline hydrolysis of phosphodiesters更多还原