Combined QM(DFT)/MM molecular dynamics simulations of the deamination of cytosine by yeast cytosine deaminase (yCD)

Extensive combined quantum mechanical (B3LYP/6‐31G*) and molecular mechanical (QM/MM) molecular dynamics simulations have been performed to elucidate the hydrolytic deamination mechanism of cytosine to uracil catalyzed by the yeast cytosine deaminase (yCD). Though cytosine has no direct binding to the zinc center, it reacts with the water molecule coordinated to zinc, and the adjacent conserved Glu64 serves as a general acid/base to shuttle protons from water to cytosine. The overall reaction consists of several proton‐transfer processes and nucleophilic attacks. A tetrahedral intermediate adduct of cytosine and water binding to zinc is identified and similar to the crystal structure of yCD with the inhibitor 2‐pyrimidinone. The rate‐determining step with the barrier of 18.0 kcal/mol in the whole catalytic cycle occurs in the process of uracil departure where the proton transfer from water to Glu64 and nucleophilic attack of the resulting hydroxide anion to C2 of the uracil ring occurs synchronously. © 2016 Wiley Periodicals, Inc.     

Deamination of the Radical Cation of the Base Moiety of 2′‐Deoxycytidine: A Theoretical Study

Five pathways leading to the deamination of cytosine (to uracil) after formation of its deprotonated radical cation are investigated in the gas phase, at the UB3LYP/6‐311G(d,p) level of theory, and in bulk aqueous solvent. The most favorable pathway involves hydrogen‐atom transfer from a water molecule to the N3 nitrogen of the deprotonated radical cation, followed by addition of the resulting hydroxyl radical to the C4 carbon of the cytosine derivative. Following protonation of the amino group (N4), the C4? N4 bond is broken with elimination of the NH₃^?+ radical and formation of a protonated uracil. The rate‐determining step of this mechanism is hydrogen‐atom transfer from a water molecule to the cytosine derivative. The associated free energy barrier is 70.2 kJ mol^?1.     

A combined ONIOM quantum chemical-molecular dynamics study of zinc-uracil bond breaking in yeast cytosine deaminase

A QM/MM method that combines ONIOM quantum chemistry and molecular dynamics is developed and applied to a step in the deamination of cytosine to uracil in yeast cytosine deaminase (yCD). A two-layer ONIOM calculation is used for the reaction complex, with an inner part treated at a high level for the chemical reaction (bond breaking) and a middle part treated at a lower level for relevant protein residues that are frozen in the quantum optimization. An outer layer (protein and solvent) is treated using MD. Configurations for the entire system are generated using MD and optimized with ONIOM. The method permits the use of high-level quantum calculations along with sufficient configurational sampling to approximate the potential of mean force for certain bond-breaking reactions. A previously proposed reaction mechanism for deamination (Sklenak, S.; Yao, L. S.; Cukier, R. I.; Yan, H. G. J. Am. Chem. Soc. 2004, 126, 14879) requires breaking the bond between a catalytic zinc and the O4 of uracil in order to permit product release. Using an ONIOM approach, direct bond cleavage was found to be energetically unfavorable. In the work presented here, the combined ONIOM MD method is used to show that the barrier for bond cleavage is small, approximately 3 kcal/mol, and, consequently, should not be the rate-limiting step in the reaction.     

A theoretical study on the catalytic mechanism of Mus musculus adenosine deaminase

The catalytic mechanism of Mus musculus adenosine deaminase (ADA) has been studied by quantum mechanics and two‐layered ONIOM calculations. Our calculations show that the previously proposed mechanism, involving His238 as the general base to activate the Zn‐bound water, has a high activation barrier of about 28 kcal/mol at the proposed rate‐determining nucleophilic addition step, and the corresponding calculated kinetic isotope effects are significantly different from the recent experimental observations. We propose a revised mechanism based on calculations, in which Glu217 serves as the general base to abstract the proton of the Zn‐bound water, and the protonated Glu217 then activates the substrate for the subsequent nucleophilic addition. The rate‐determining step is the proton transfer from Zn‐OH to 6‐NH₂ of the tetrahedral intermediate, in which His238 serves as a proton shuttle for the proton transfer. The calculated kinetic isotope effects agree well with the experimental data, and calculated activation energy is also consistent with the experimental reaction rate. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

Ring cleavage of aziridines by difluoroamine: mechanistic insights from ab initio and DFT study

cis-2,3-Dimethylaziridine reacts with difluoroamine to give the corresponding alkene and nitrogen with retention of configuration. We have carried out a DFT study of this reaction to clarify the reaction mechanism by considering a multistep reaction pathway with possible intermediacy of several three- and four-membered cyclic intermediates and transition states (TSs). The energetics of this reaction shows that the reaction takes place in four steps including a three-membered azamine intermediate. Both the energetics and the stereochemical outcome of this reaction rule out the formation of a four-membered diazetine intermediate during the reaction. Although the first N-N bond formation step is rate determining, the final step, asynchronous concerted cleavage of the azamine intermediate, explains the stereochemistry of this reaction. The asynchronous nature of the final step makes the reaction Woodward-Hoffmann allowed, as reported by Yamabe and Minato (J. Phys. Chem. A 2001, 105, 7281). Computations at HF and MP2 levels confirm the same trends in energetics. Single point energy computations at B3LYP, MP2, and QCISD levels with the 6-311++G(d,p) and cc-pVTZ basis sets show that the larger basis sets predict higher free energies of activation and less negative free energies of reaction. Intrinsic reaction coordinate (IRC) analyses reveal the asynchronous nature of the first and the last steps of the reaction. The deamination of trans-2,3-dimethylaziridine was shown to follow a course of reaction similar to that of the cis isomer.     

Anilinolysis of Picryl Benzoate Derivatives in Methanol: Reactivity,Regioselectivity, Kinetics,and Mechanism

The reaction of picryl benzoate derivatives 1a–g with aniline in methanol proceeds through CO? O and Ar? O bond cleavage pathways. Furthermore, the reactivity of these esters toward anilinolysis is correlated to the energy gap between highest occupied molecular orbital aniline and lowest unoccupied molecular orbital of each ester. The regioselectivity of acyl? oxygen versus aryl? oxygen cleavage is also discussed. The overall rate constants k_tot split into k_{CO? O} (the rate constant of acyl‐oxygen cleavage) and k_{Ar? O} (rate constant of aryl‐oxygen cleavage). The CO? O bond cleavage advances through a stepwise mechanism in which the formation of the tetrahedral intermediate is the rate‐determining step. The Ar? O bond cleavage continues through a S_NAr mechanism in which the departure of the leaving group from the Meisenheimer complex occurs rapidly after its formation in the rate‐determining step.     

Dinuclear Zn(II) complex catalyzed phosphodiester cleavage proceeds via a concerted mechanism: a density functional theory study

Density functional theory (DFT) calculations were used to study the mechanism for the cleavage reaction of the RNA analogue HpPNP (HpPNP = 2-hydroxypropyl-4-nitrophenyl phosphate) catalyzed by the dinuclear Zn(II) complex of 1,3-bis(1,4,7-triazacyclonon-1-yl)-2-hydroxypropane (Zn(2)(L(2)O)). We present a binding mode in which each terminal phosphoryl oxygen atom binds to one zinc center, respectively, and the nucleophilic 2-hydroxypropyl group coordinates to one of the zinc ions, while the hydroxide from deprotonation of a water molecule coordinates to the other zinc ion. Our calculations found a concerted mechanism for the HpPNP cleavage with a 16.5 kcal/mol reaction barrier. An alternative proposed stepwise mechanism through a pentavalent oxyphosphorane dianion reaction intermediate for the HpPNP cleavage was found to be less feasible with a significantly higher energy barrier. In this stepwise mechanism, the deprotonation of the nucleophilic 2-hydroxypropyl group is accompanied with nucleophilic attack in the rate-determining step. Calculations of the nucleophile (18)O kinetic isotope effect (KIE) and leaving (18)O KIE for the concerted mechanism are in reasonably good agreement with the experimental values. Our results indicate a specific-base catalysis mechanism takes place in which the deprotonation of the nucleophilic 2-hydroxypropyl group occurs in a pre-equilibrium step followed by a nucleophilic attack on the phosphorus center. Detailed comparison of the geometric and electronic structure for the HpPNP cleavage reaction mechanisms in the presence/absence of catalyst revealed that the catalyst significantly altered the determining-step transition state to become far more associative or tight, that is, bond formation to the nucleophile was remarkably more advanced than leaving group bond fission in the catalyzed mechanism. Our results are consistent with and provide a reliable interpretation for the experimental observations that suggest the reaction occurs by a concerted mechanism (see Humphry, T.; Iyer, S.; Iranzo, O.; Morrow, J. R.; Richard, J. P.; Paneth, P.; Hengge, A. C. J. Am. Chem. Soc. 2008, 130, 17858-17866) and has a specific-base catalysis character (see Yang, M.-Y.; Iranzo, O.; Richard, J. P.; Morrow, J. R. J. Am. Chem. Soc. 2005, 127, 1064-1065).     

Zinc(II) complexes with intramolecular amide oxygen coordination as models of metalloamidases

Polydentate ligands (6-R1-2-pyridylmethyl)-R2(R1= NHCOtBu, R2= bis(2-pyridylmethyl)amine L1, bis(2-(methylthio)ethyl)amine L2 and N(CH2CH2)2S L3) form mononuclear zinc(II) complexes with intramolecular amide oxygen coordination and a range of coordination environments. Thus, the reaction of Zn(ClO4)2.6H2O with L1-3 in acetonitrile affords [(L)Zn](ClO4)2(L=L1, 1; L2, 2) and [(L3)Zn(H2O)(NCCH3)](ClO4)2 3. The simultaneous amide/water binding in resembles the motif that has been proposed to be involved in the double substrate/nucleophile Lewis acidic activation and positioning mechanism of amide bond hydrolysis in metallopeptidases. X-ray diffraction, 1H and 13C NMR and IR data suggests that the strength of amide oxygen coordination follows the trend 1>2 >3. L1-3 and undergo cleavage of the tert-butylamide upon addition of Me4NOH.5H2O (1 equiv.) in methanol at 50(1)degrees C. The rate of amide cleavage follows the order 1> 2> 3, L1-3. The extent by which the amide cleavage reaction is accelerated in 1-3 relative to the free ligands, L1-3, is correlated with the strength of amide oxygen binding and Lewis acidity of the zinc(II) centre in deduced from the X-ray, NMR and IR studies.     

Proton catalyzed hydrolytic deamination of cytosine: a computational study

Two pathways involving proton catalyzed hydrolytic deamination of cytosine (to uracil) are investigated at the PCM-corrected B3LYP/6-311G(d,p) level of theory, in the presence of an additional catalyzing water molecule. It is concluded that the pathway involving initial protonation at nitrogen in position 3 of the ring, followed by water addition at C4 and proton transfer to the amino group, is a likely route to hydrolytic deamination. The rate determining step is the addition of water to the cytosine, with a calculated free energy barrier in aqueous solution of ΔG ^≠=140 kJ/mol. The current mechanism provides a lower barrier to deamination than previous work based on OH ^? catalyzed reactions, and lies closer to the experimental barrier derived from rate constants (E _a = 117  ±  4 kJ/mol).     

Ferric superoxide and ferric hydroxide are used in the catalytic mechanism of hydroxyethylphosphonate dioxygenase: a density functional theory investigation

Hydroxyethylphosphonate dioxygenase (HEPD) is a mononuclear nonheme iron enzyme that utilizes an O(2) molecule to cleave a C-C bond in 2-hydroxyethylphosphonate and produce hydroxymethylphosphonate (HMP) and formic acid. Density functional theory calculations were performed on an enzyme active-site model of HEPD to understand its catalytic mechanism. The reaction starts with H-abstraction from the C2 position of 2-HEP by a ferric superoxide-type (Fe(III)-OO(?-)) intermediate, in a similar manner to the H-abstraction in the reaction of the dinuclear iron enzyme myo-inositol oxygenase. The resultant Fe(II)-OOH intermediate may follow either a hydroperoxylation or hydroxylation pathway, the former process being energetically more favorable. In the hydroperoxylation pathway, a ferrous-alkylhydroperoxo intermediate is formed, and then its O-O bond is homolytically cleaved to yield a complex of ferric hydroxide with a gem-diol radical. Subsequent C-C bond cleavage within the gem-diol leads to formation of an R-CH(2)(?) species and one of the two products (i.e., formic acid). The R-CH(2)(?) then intramolecularly forms a C-O bond with the ferric hydroxide to provide the other product, HMP. The overall reaction pathway does not require the use of a high-valent ferryl intermediate but does require ferric superoxide and ferric hydroxide intermediates.     

Mechanisms for the deamination reaction of cytosine with H2O/OH(-) and 2H2O/OH(-): a computational study

Mechanisms for the deamination reaction of cytosine with H 2O/OH (-) and 2H 2O/OH (-) to produce uracil were investigated using ab initio calculations. Optimized geometries of reactants, transition states, intermediates, and products were determined at MP2 and B3LYP using the 6-31G(d) basis set and at B3LYP/6-31+G(d) levels of theory. Single point energies were also determined at MP2/G3MP2Large and G3MP2 levels of theory. Thermodynamic properties (Delta E, Delta H, and Delta G), activation energies, enthalpies, and free energies of activation were calculated for each reaction pathway investigated. Intrinsic reaction coordinate (IRC) analysis was performed to characterize the transition states on the potential energy surface. Seven pathways for the deamination reaction were found. All pathways produce an initial tetrahedral intermediate followed by several conformational changes. The final intermediate for all pathways dissociates to product via a 1-3 proton shift. The activation energy for the rate-determining step, the formation of the tetrahedral intermediate for pathway D, the only pathway that can lead to uracil, is 115.3 kJ mol (-1) at the G3MP2 level of theory, in excellent agreement with the experimental value (117 +/- 4 kJ mol (-1)).     

Oxidative behavior and relative reactivities of some unsaturated compounds towards hexachloroiridate(IV) in perchloric acid medium

The kinetics of the oxidation of styrene, cinnamic acid, and some of their substituted derivatives by hexachloroiridate(IV) in dimethyl formamide–water mixtures and in the presence of perchloric acid have been investigated. The reactions appear to proceed via the formation of an unstable intermediate 1:1 complex between iridium(IV) and the substrate, followed by the decomposition of the complex in the rate‐determining step. Correlation with σ yielded ρ values of ?4.0 and ?3.5 which suggests the formation of a cationic intermediate in the rate‐determining step of the reaction. Subsequent cleavage of the carbon–carbon bond yielded the product aldehydes. Thermodynamic and activation parameters associated with the equilibrium and the rate‐determining steps were also evaluated. © 2002 Wiley Periodicals, Inc. Int J Chem Kinet 34: 411–417, 2002     

Mechanism of Ketone Allylation with Allylboronates as Catalyzed by Zinc Compounds: A DFT Study

The mechanism of the allylation reaction between 4‐chloroacetophenone and pinacol allylboronates catalyzed by ZnEt₂ with alcohols was investigated using density functional theory (DFT) at the M05‐2X/6‐311++G(d,p) level. The calculations reveal that the reaction prefers to proceed through a double γ‐addition stepwise reaction mechanism rather than a Lewis acid‐catalyzed concerted one. The intermediate with a four‐coordinated boron center, which is formed through proton transfer from EtOH to the ethyl group of ZnEt₂ mediated by the boron center, is the active species and an entrance for the catalytic cycle. The latter is composed of three elementary steps: 1) boron to zinc transmetalation leading to the formation of allylzincate species, 2) electrophilic addition of ketone to allylzincate species, and 3) generation of the final product with recovery of the catalyst. The boron to zinc transmetalation step has the largest energy barrier of 61.0 kJ mol^?1 and is predicted to be the rate‐determining step. The calculations indicate that the additive EtOH plays important roles both in lowering the activation free energy for the formation of the four‐coordinated boron active intermediate and in transforming the low catalytic activity ZnEt₂ into high activity zinc alkoxide species. The alcohols with a less sterically encumbering R group might be the effective additives. The substituted groups on the allylboronates might primarily affect the boron to zinc transmetalation, and the allylboronates with substituents on the C_γ atom is poor in reactivity. The comparison of the catalytic effect between the zinc compounds investigated suggest that Zn(OEt)₂, Zn(OH)₂, and ZnF₂ exhibit higher catalytic efficiency for the boron to zinc transmetalation due to the activation of the B? C_α bond through orbital interactions between the p orbitals of the EtO, OH, F groups and the empty p orbital of the boron center.     

Catalytic mechanism of guanine deaminase: an ONIOM and molecular dynamics study

The catalytic mechanism of Bacillus subtilis guanine deaminase (bGD), a Zn metalloenzyme, has been investigated by a combination of quantum mechanical calculations using the multilayered ONIOM method and molecular dynamics simulations. In contrast to a previously proposed catalytic mechanism, which requires the bound guanine to assume a rare tautomeric state, the ONIOM calculations showed that the active-site residues of the enzyme do not affect the tautomeric state of guanine, and consequently the bound guanine is a tautomer that is the most abundant in aqueous solution. Two residues, Glutamate 55 and Aspartate 114, were found to play important roles in proton shuttling in the reaction. The proposed reaction path is initiated by proton transfer from a Zn-bound water to protonate Asp114. This process may be quite complex and rather dynamic in nature, as revealed by the molecular dynamics (MD) simulations, whereby another water may bridge the Zn-bound water and Asp114, which then is eliminated by positioning of guanine in the active site. The binding of guanine stabilizes protonated Asp114 by hydrogen bond formation. Asp114 can then transfer its proton to the N3 of the bound guanine, facilitating the nucleophilic attack on C2 of the guanine by the Zn-bound hydroxide to form a tetrahedral intermediate. This occurs with a rather low barrier. Glu55 then transfers a proton from the Zn-hydroxide to the amino group of the reaction intermediate and, at this point, the C2-N2 bond has lengthened by 0.2 A compared to guanine, making C2-N2 bond cleavage more facile. The C2-N2 bond breaks forming ammonia, with an energy barrier of approximately 8.8 kcal/mol. Ammonia leaves the active site, and xanthine is freed by the cleavage of the Zn-O2 bond, with a barrier approximately 8.4 kcal/mol. Along this reaction path, the highest barrier comes from C2-N2 bond cleavage, while the barrier from the cleavage of the Zn-O2 bond is slightly smaller. The Zn-O2 bond can be broken without the assistance of water during the release of xanthine.     

金属、配体和溶剂之间的协同性：基于密度泛函理论研究含双锌离子配合物催化磷酸二酯裂解的反应机理

Density functional theory (DFT)-Tao-Perdew-Staroverov-Scuseria (TPSS) functional calculations on dizinc complex-mediated phosphodiester cleavage indicate a general base catalytic mechanism. 2-hydroxylpropyl-4-nitrophenyl phosphate (HPNP) favors the bridging of two Zn ions by the formation of two coordination bonds between terminal phosphate oxygens and Zn ions. The Zn-bound hydroxide deprotonates the hydroxyl on the side chain of HPNP and consequently the alkoxide is stabilized by coordination to a Zn ion and a hydrogen-bond to Zn-bound water. A water molecule is tightly bound to two amino protons in the bis(1,4,7-triazacyclononane) ligand and this determines the orientation of HPNP during a nucleophilic attack to form a trigonal bipyramidal PO5 intermediate and it also weakens the bond between phosphorus and the phenolate, which makes the leaving of the latter easier. The phenolate formed after the collapse of the five-coordinated phosphorus intermediate easily coordinates to a Zn ion. Surprisingly, the stabilizing solvent effect for the transition state after the formation of the PO5 intermediate is much stronger (at least 42 kJ·mol-1) than that of all other species as they have solvation energies that fluctuate around 12.6 kJ·mol-1. Thus, the overall free energy barrier for this reaction after reactant-binding and before product release is about 17.0 kJ·mol -1, which is too low to be rate-determining. The rate-determining step is very likely part of the release process of the products. Based on various calculations, we discuss possible reasons for the different catalytic efficiencies of the dizinc complex and the enzymes.     

Compared behavior of 5-deoxy-5-iodo-D-xylo- and L-arabinofuranosides in the reductive elimination reaction: a strong dependence on structural parameters and on the presence of Zn2+. A combined experimental and theoretical investigation

In the framework of a project devoted to the chemical transformation of monosaccharides from hemicelluloses into higher added value materials, the zinc-induced reductive elimination from 5-deoxy-5-iodo derivatives of D-xylose and L-arabinose was carried out. This study gave us the opportunity to observe surprising behaviors. In particular, the reaction strongly depends on structural parameters (protecting group pattern, configuration at C-4) and on the presence of Zn2+ ions. Collaterally with the experimental work, water solvent PCM HF-DFT (MPW1K/LANL2DZ) computations were performed to obtain insight into the mechanism for the reductive part of the reaction sequence. Without Zn2+, the zinc insertion reaction was found to proceed through a concerted but non-synchronous process involving a relatively large energy barrier (32 kcal mol-1) that directly leads to the presumed organozinc intermediate. In the presence of Zn2+, a three-step mechanism was identified in which the cation coordinates the anomeric and ring oxygen atoms and also the sugar iodine atom, causing an activating effect on the zinc insertion process by facilitating the homolytic rupture of the C-I bond. Complexes between zinc and Zn2+ bound carbohydrates were characterized with large stabilization energies, suggesting that Zn2+ might enhance the affinity of the organic compound with the zinc metal surface.     

Oxidative C-C bond cleavage of primary alcohols and vicinal diols catalyzed by H5PV2Mo10O40 by an electron transfer and oxygen transfer reaction mechanism

Primary alcohols such as 1-butanol were oxidized by the H5PV2Mo10O40 polyoxometalate in an atypical manner. Instead of C-H bond activation leading to the formation of butanal and butanoic acid, C-C bond cleavage took place leading to the formation of propanal and formaldehyde as initial products. The latter reacted with the excess 1-butanol present to yield butylformate and butylpropanate in additional oxidative transformations. Kinetic studies including measurement of kinetic isotope effects, labeling studies with 18O labeled H5PV2Mo10O40, and observation of a prerate determining step intermediate by 13C NMR leads to the formulation of a reaction mechanism based on electron transfer from the substrate to the polyoxometalate and oxygen transfer from the reduced polyoxometalate to the organic substrate. It was also shown that vicinal diols such as 1,2-ethanediol apparently react by a similar reaction mechanism.     

Multiple isotope effect study of the acid-catalyzed hydrolysis of formamide

Multiple isotope effects were measured at the reactive center of formamide during acid-catalyzed hydrolysis in water at 25 degrees C. The mechanism involves a rapid pre-equilibrium protonation of the carbonyl oxygen, followed by the formation of at least one tetrahedral intermediate, which does not appreciably exchange its carbonyl oxygen with the solvent (kh/kex = 55). The pKa for formamide was determined by 15N NMR and found to be about -2.0. The formyl-hydrogen kinetic isotope effect (KIE) is indicative of a transition state that is highly tetrahedral (Dkobs = 0.79); the carbonyl-carbon KIE (13kobs = 1.031) is in agreement with this conclusion. The small leaving-nitrogen KIE (15kobs = 1.0050) is consistent with some step prior to breaking the C-N bond as rate-determining. The carbonyl-oxygen KIE (18kobs = 0.996) points to attack of water as the rate-determining step. On the basis of these results, a mechanism is proposed in which attachment of the nucleophile to a protonated formamide molecule is rate determining.     

Reaction mechanism of uracil bromination by HBrO: a new way to generate the enol-keto form of 5-bromouracil

Knowledge on the uracil bromination reaction is helpful for understanding the origin of the mutagenicity of 5-bromouracil (BrU). To get more details about this reaction, we explore the corresponding reaction mechanism by theoretical method. A total of seven pathways were studied for this purpose. The diketo form of BrU is observed as the main product in these pathways, which agrees well with experimental results. The most energy-favorable reaction pathway is found to be that Br and OH attacked the opposite sides of uracil. The reaction intermediate reported in the experiment is predicted to be reasonably stable. In the following step, a dehydration process occurs between H11 and O13-H14 when there are no explicit H(2)O taking part. However, when there are explicit water molecules in the environment, explicit H(2)O will lower the reaction barrier in the formation of reaction intermediates and the final product BrU. A proton-transfer process from C5 to O10 is facilitated by explicit H(2)O, which results in enol-keto form intermediate of this modified base (defined as BrU*). These results indicate a new way to generate the enol-keto form of BrU.     

Theoretical study on the hydrolytic deamination reaction mechanism of guanine and (H2O)n

The hydrolytic deamination reaction mechanism of guanine (G) and (H₂O)n (n = 1–4) has been theoretically investigated, including solvent effects at the B3LYP/6-31G (d,p) and MP2/6-311G (d,p) levels. The results show that the hydrolytic deamination reaction of G involves two steps. In the first step, a tetra-coordinated intermediate is generated by a hydrolysis reaction, and then deamination reaction is carried out. In the second step, the final products form via a hydrogen transfer. For the hydrolytic deamination reaction of guanine and 3H₂O or 4H₂O, only two molecules of water participate in the reaction, and other water molecule make the transition state more stable as a catalyst. The study on the potential energy surface shows that the deamination reaction of G and nH₂O does not take place because of a higher barrier for the opening system, which is in agreement with the experimental result.