Study On Kinetic Isotope Effect And Mechanism In The Process Of Nucleic Acid Demethylation

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.