【作者】 王颖；

【导师】 张建宇； 张秀阁；

【作者基本信息】 天津大学 ， 药学， 2021， 硕士

【摘要】 甲基化是真核细胞DNA中常见且广泛的修饰方法,占RNA化学修饰的三分之二。N6-甲基腺苷（m⁶A）于1970年发现,在遗传过程中起不可或缺的作用。核酸甲基化可以在依赖Fe（II）和α-KG的大肠杆菌双加氧酶ALKB和FTO的催化下逆转。我们利用液相质谱法（LC-MS）对酶甲基化体系和化学去甲基化的动力学同位素效应（KIE）和机制进行了详细研究。我们获得了足够量的FTO和ALKB蛋白来研究酶促去甲基化动态过程中的KIE。以m⁶A为底物,竞争性底物上甲基化位点的三个H原子被D原子取代。通过LC-MS检测反应中m⁶A-D3与m⁶A含量的比例变化,并基于此分析过渡态。根据公式k _L/k _H=ln（1-f _L）/ln[（1-f _L）R_S/R_OS]和ln(R_S/R_OS)=（1/KIE-1）ln（1-f _L）处理拟合数据后,FTO和ALKB的KIE值分别为1.084±0.007和1.050±0.004,相关系数均大于0.98。根据这两组数值范围,初步确定m⁶A-D3的FTO和ALKB的脱甲基过程包含二级同位素效应,即在决速步中没有发生同位素参与的化学键断裂,但杂化轨道重新杂化或减弱。FTO和ALKB的KIE值略有不同,但具有相同的决速步:从中间体hm⁶A水解为甲醛分子和腺嘌呤的过程。作为ALKB和FTO的新特征,这为将来深入研究脱甲基机制提供了新的方向。同时,我们还对化学脱甲基系统进行了详细的机理研究,为酶系统提供了分析参考和模型。已知由过氧化氢和碳酸氢铵组成的氧化系统可以完成化学去甲基反应。硫酸亚铁铵（FAS）可以与过氧化氢形成Fenton试剂,以提高氧化速率。但是具体的机制和决速步尚待探索。我们使用LC-MS研究了硫酸亚铁铵浓度对化学去甲基化系统KIE的影响。随着亚铁离子浓度的增加,KIE从1.377±0.020降低至1.120±0.016,我们认为是一级同位素效应和逆反二级同位素效应的组合,即KIE_obs=1°KIE*（2°KIE）²。通过分析KIE趋势,我们认为初始拔氢阶段是决速步,而且hm⁶A形成过程中有紧密的过渡态结构。KIE与过渡态周围空间的拥挤程度直接相关,羟基自由基浓度的增加限制了过渡态的平面外弯曲振动。我们计算得出该反应中1°KIE和逆反2°KIE的范围分别为2.000².290和0.700⁰.830。我们还捕捉到了反应过程中的中间体hm⁶A及其氘代化合物,并提出了更详细的化学去甲基化机制。FAS浓度依赖的KIE从新颖的角度解释了化学去甲基化的过渡状态,中间体的捕获使自由基的机理更加具体和准确,这也为酶催化脱甲基化提供了新的研究模型。更多还原

【Abstract】 Methylation is a common and extensive modification method in eukaryotic DNA,and it also accounts for two-thirds of RNA chemical modification.N6-methyladenosine（m⁶A）was discovered in 1970 and plays an indispensable role in the genetic process.Nucleic acid methylation can be reversed under the catalysis of E.coli dioxygenase ALKB and FTO,which rely on Fe（II）andα-KG.We used liquid-phase mass spectrometry（LC-MS）to study the kinetic isotope effect（KIE）and mechanism of the enzyme methylation system in detail.We have obtained enough FTO and ALKB proteins to study KIE in the dynamic process of enzymatic demethylation.With m⁶A as the substrate,the three H atoms at the methylation sites on the competitive substrate were replaced by D atoms.The change in the content ratio of m⁶A-D3 to m⁶A in the reaction was detected by LC-MS,and the transition state was analyzed based on this.After processing data according to the formula k _L/k _H=ln（1-f _L）/ln[（1-f _L）R_S/R_OS]and ln(R_S/R_OS)=（1/KIE-1）ln（1-f _L）,the KIE values of FTO and ALKB are 1.084±0.007 and 1.050±0.004,respectively,and the correlation coefficients are both greater than 0.98.According to these two sets of numerical ranges,it is preliminarily determined that the demethylation process of FTO and ALKB of m⁶A-D₃ contained secondary isotope effects.In the rate determining step,the chemical bonds connected by the deuterium atoms are not broken,but the hybrid orbitals are re-hybridized or weakened.The KIE values of FTO and ALKB are slightly different,but they have the same decisive step:the process of hydrolysis from the intermediate hm⁶A to formaldehyde molecules and adenine.As a new feature of ALKB and FTO,this provides a new direction for the in-depth study of the demethylation mechanism in the future.At the same time,we have also carried out a detailed mechanism study on the chemical demethylation system,which provides an analysis reference and model for the enzyme system.It is known that an oxidation system composed of hydrogen peroxide and ammonium bicarbonate can complete a chemical demethylation reaction.Ferrous ammonium sulfate（FAS）can form Fenton reagent with hydrogen peroxide to increase the oxidation rate.However,the specific mechanism and decisive steps have yet to be explored.We used LC-MS to study the effect of the concentration of ferrous ammonium sulfate on the KIE of the chemical demethylation system.As the concentration of ferrous ions increases,KIE decreases from 1.377±0.020 to 1.120±0.016.We speculate it is a combination of primary isotope effect and reverse secondary isotope effect,namely KIE_obs=1°KIE*（2°KIE）².By analyzing the KIE trend,we believe that the initial hydrogen removal stage is a rate determining step,,and there is a tight transition state structure during the formation of hm⁶A.KIE is directly related to the crowding degree of the space around the transition state.The increase in the concentration of hydroxyl radicals limits the out-of-plane bending vibration of the transition state.We calculated that the ranges of 1°KIE and reverse 2°KIE in this reaction were 2.000～2.290 and 0.700～0.830,respectively.We also captured the intermediate hm⁶A and its deuterated compounds during the reaction,and proposed a more detailed chemical demethylation mechanism.The FAS concentration-dependent KIE explains the transition state of chemical demethylation from a novel perspective.The capture of intermediates makes the mechanism of free radicals more specific and accurate,which also provides a new research model for enzyme-catalyzed demethylation.更多还原