Catalytic mechanism of yeast cytosine deaminase: an ONIOM computational study

The complete path for the deamination reaction catalyzed by yeast cytosine deaminase (yCD), a zinc metalloenzyme of significant biomedical interest, has been investigated using the ONIOM method. Cytosine deamination proceeds via a sequential mechanism involving the protonation of N(3), the nucleophilic attack of C(4) by the zinc-coordinated hydroxide, and the cleavage of the C(4)-N(4) bond. The last step is the rate determining step for the generation of the zinc bound uracil. Uracil is liberated from the Zn atom by an oxygen exchange mechanism that involves the formation of a gem-diol intermediate from the Zn bound uracil and a water molecule, the C(4)-O(Zn) cleavage, and the regeneration of the Zn-coordinated water. The rate determining step in the oxygen exchange is the formation of the gem-diol intermediate, which is also the rate determining step for the overall yCD-catalyzed deamination reaction.     

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.     

Ab initio ONIOM-molecular dynamics (MD) study on the deamination reaction by cytidine deaminase

We applied the ONIOM-molecular dynamics (MD) method to the hydrolytic deamination of cytidine by cytidine deaminase, which is an essential step of the activation process of the anticancer drug inside the human body. The direct MD simulations were performed for the realistic model of cytidine deaminase by calculating the energy and its gradient by the ab initio ONIOM method on the fly. The ONIOM-MD calculations including the thermal motion show that the neighboring amino acid residue is an important factor of the environmental effects and significantly affects not only the geometry and energy of the substrate trapped in the pocket of the active site but also the elementary step of the catalytic reaction. We successfully simulate the second half of the catalytic cycle, which has been considered to involve the rate-determining step, and reveal that the rate-determining step is the release of the NH3 molecule.     

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.     

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.     

PHOTOCYCLOADDITION OF ACETONE TO URACIL AND CYTOSINE

Abstract— Ultraviolet irradiation (Λ > 280 nm) of uracil in aqueous acetone (1:1) produces cyclobutane uracil dimer and uracil-acetone addition product. The addition product is identified as an oxetane. The same product is also obtained from cytosine irradiated under the same conditions. The cytosine-acetone oxetane apparently undergoes deamination readily. Irradiation (Λ= 265 nm) of the oxetane in aqueous solution produces acetone and uracil with a quantum efficiency of 0–16.     

Site-specific transition of cytosine to uracil via reversible DNA photoligation

We report that deamination coupled with 5-carboxyvinyldeoxyuridine-mediated photobranching causes the heat-induced transition of cytosine to uracil with high efficiency without any side reaction.     

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).     

Hydride transfer catalyzed by xylose isomerase: mechanism and quantum effects

We have applied molecular dynamics umbrella-sampling simulation and ensemble-averaged variational transition state theory with multidimensional tunneling (EA-VTST/MT) to calculate the reaction rate of xylose-to- xylulose isomerization catalyzed by xylose isomerase in the presence of two Mg2+ ions. The calculations include determination of the free energy of activation profile and ensemble averaging in the transmission coefficient. The potential energy function is approximated by a combined QM/MM/SVB method involving PM3 for the quantum mechanical (QM) subsystem, CHARMM22 and TIP3P for the molecular mechanical (MM) environment, and a simple valence bond (SVB) local function of two bond distances for the hydride transfer reaction. The simulation confirms the essential features of a mechanism postulated on the basis of kinetics and X-ray data by Whitlow et al. (Whitlow, M.; Howard, A. J.; Finzel, B. C.; Poulos, T. L.; Winborne, E.; Gilliland, G. L. Proteins 1991, 9, 153) and Ringe, Petsko, and coworkers (Labie, A.; Allen, K.-N.; Petsko, G. A.; Ringe, D. Biochemistry 1994, 33, 5469). This mechanism involves a rate-determining 1,2-hydride shift with prior and post proton transfers. Inclusion of quantum mechanical vibrational energy is important for computing the free energy of activation, and quantum mechanical tunneling effects are essential for computing kinetic isotope effects (KIEs). It is found that 85% of the reaction proceeds by tunneling and 15% by overbarrier events. The computed KIE for the ratio of hydride to deuteride transfer is in good agreement with the experimental results. The molecular dynamics simulations reveal that proton and hydride transfer reactions are assisted by breathing motions of the mobile Mg2+ ion in the active site, providing evidence for concerted motion of Mg2+ during the hydride transfer step.     

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)).     

Theoretical study of the catalytic mechanism and metal-ion dependence of peptide deformylase

The reaction pathway of deformylation catalyzed by E. coli peptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, and cleavage of the C-N bond. The first step is rate-determining for the deformylation, which occurs through a pentacoordinated metal center. The estimated activation energies with the ONIOM method are about 23.0, 15.0, and 14.9 kcal/mol for Zn-, Ni-, and Fe-PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the preference for metal coordination geometry exerts a significant influence on the catalytic activity of PDFs by affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of the PDFs.     

Can cytosine, thymine and uracil be formed in interstellar regions? A theoretical study

This theoretical study investigates possible synthetic routes to cytosine, uracil and thymine in the gas phase from precursor molecules that have been detected in interstellar media. Studies at the CCSD(T)/6-311++G(d,p)//B3LYP/6-311++G(d,p) level of theory suggest that: The reactions between :CCCNH and :CCCO with monosolvated urea may constitute viable interstellar syntheses of cytosine and uracil. No low energy equilibration between cytosine and uracil has been demonstrated. The interaction of :CH(2) with the 5 C-H bond of uracil may form thymine in an energetically favourable reaction, but competing reactions where :CH(2) reacts with double bonds and other CH and NH bonds of uracil, reduce the effectiveness of this synthesis. The reaction between the hydrated propional enolate anion and isocyanic acid may produce thymine, in a reaction sequence where ΔG(reaction)(298 K) is -22 kJ mol(-1) and the maximum energy requirement (barrier to the first transition state) is only 47 kJ mol(-1).     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

Reactions of pyrimidine nucleosides with aqueous alkalies: kinetics and mechanisms

Kinetics for the reactions of various cytosine and uracil nucleosides and their alkyl derivatives with aqueous sodium hydroxide have been studied by liquid chromatography. Blocking of the glycosyl hydroxyl groups with alkyl groups and changes in the glycon moiety configuration have been observed to exert only moderate effects on the rate of deamination of cytosine nucleosides. Methylation of the 4-amino group retards deamination considerably, while a methyl substituent at C5 is rate accelerating and at C6 only moderately rate retarding. These findings have been accounted for by a mechanism involving a rate limiting bimolecular displacement of the 4-amino group by a hydroxide ion. Analogous comparisons with uracil nucleosides suggest that the decomposition of uridine is initiated by an intermolecular attack of hydroxide ion on the C5 atom of the base moiety. In contrast, beta-D-arabino- and beta-D-lyxo-furanosyl derivatives appear to be cleaved via an intramolecular nucleophilic attack of the ionized 2'-hydroxyl group.     

Solvent effects on the asymmetric Diels–Alder reaction between cyclopentadiene and (−)-menthyl acrylate revisited with the three-layer hybrid local self-consistent field/molecular mechanics/self-consistent reaction field method

The construction of the three-layer hybrid local self-consistent field/molecular mechanics/self-consistent reaction field method is detailed. This method is specifically devoted to the study of the reactivity of large chemical systems in solution. The solvent, modeled by a polarizable continuum, surrounds the whole solute molecule. Solute–solvent interactions are taken into account by means of the self-consistent reaction field approach. The solute system is treated by both quantum and molecular mechanics, the former being principally applied to the reactive part, i.e., the part undertaking bond forming or breaking, the latter being reserved for the ancillary encumbering groups. The connection between the molecular mechanics and the quantum mechanics part is accomplished by a strictly localized bond orbital that remains frozen within the local self-consistent field framework. As a test system, the asymmetric Diels–Alder reaction between cyclopentadiene and (–)-menthyl acrylate is studied for the first time with steric interactions and electrostatic solvent effects taken into account simultaneously. The results indicate that the coupling of both interactions leads to conclusions that could not have been guessed from separate calculations.Proceedings of the 11th International Congress of Quantum chemistry satellite meeting in honour of Jean-Louis Rivail     

S(H)2 reaction vs hydrogen abstraction/expulsion in methyl radical-methylsilane reactions: effects of prereactive complex formation

A quantum chemical study has been undertaken to elucidate the cause of the recently observed S(H)2 reaction between the deuterated methyl radical (*CD3) and methylsilane (SiD3CH3) following the photolysis of CD3I. [Komaguchi, K.; Norberg, D.; Nakazawa, N.; Shiotani, M.; Persson, P.; Lunell, S. Chem. Phys. Lett. 2005, 410, 1-5.] It is found that the backside S(H)2 mechanism may proceed favorably for C-Si-C angles deviating with up to 40 degrees from linearity. The competitive hydrogen abstraction reaction is predicted to be active in the range of 90 degrees 相似文献   

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).     

Force‐Induced Reversal of β‐Eliminations: Stressed Disulfide Bonds in Alkaline Solution

Understanding the impact of tensile forces on disulfide bond cleavage is not only crucial to the breaking of cross‐linkers in vulcanized materials such as strained rubber, but also to the regulation of protein activity by disulfide switches. By using ab initio simulations in the condensed phase, we investigated the response of disulfide cleavage by β‐elimination to mechanical stress. We reveal that the rate‐determining first step of the thermal reaction, which is the abstraction of the β‐proton, is insensitive to external forces. However, forces larger than about 1 nN were found to reshape the free‐energy landscape of the reaction so dramatically that a second channel is created, where the order of the reaction steps is reversed, turning β‐deprotonation into a barrier‐free follow‐up process to C?S cleavage. This transforms a slow and force‐independent process with second‐order kinetics into a unimolecular reaction that is greatly accelerated by mechanical forces.     

Effect of Al?H···H?O dihydrogen bond on the reaction between diphenylmethanol and pyrazolate‐bridged dialuminum complex. An ONIOM DFT/AM1 study

To elucidate the nature of the Al? H···H? O dihydrogen bond and its effect on the reaction between diphenylmethanol and pyrazolate‐bridged dialuminum complex, a theoretical study was carried out using the ONIOM(B3LYP/6‐31+G(d,p):AM1) method. Calculations indicate that this reaction is a two‐step process. The first step is nucleophilic addition and the resulting intermediate is stabilized by an Al? H···H? O dihydrogen bond. Topology analyses based on the “atoms‐in‐molecules” theory show that the Al? H···H? O dihydrogen bond in dialuminum intermediate is stronger than normal hydrogen bond. This step is not barrierless, which is contrary to the result predicted by using simplified model. The second step, eliminating a molecule of dihydrogen, requires an activation free energy of 9.9 kcal/mol in gas phase, which implies the simplified model underestimates the energy barrier of this elimination step. ONIOM calculations also show that, using the simplified model without zero‐point energy correction, the dihydrogen bonding strength has been underestimated and unreliable results have been obtained. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2008     

Ab initio studies on the thermolysis of azetidine

The reaction mechanism of the thermolysis of azetidine to form ethylene and methylen-imine has been studied by ab initio SCF MO method at STO--3G and 3-21G levels. Two possible step-wise pathways are explored. One is the breaking of C--C bond as the first step, while the other is thebreaking of C--N bond. All the stationary points on the potential energy surface (PES) are fully optimiz-ed. MP2 / 3-21G single point calculations on all stationary points and MCSCF / STO-3G computationsfor some stationary points are also carried out. The calculations indicate that azetidine decomposesvia biradicaloid intermediates and the cleavage of C--N bond is preferable to that of C--C bond.