This study uses a variety of computational models and detailed, systematic potential energy surface scans to examine the hydrolysis of 2'-deoxyuridine. First, the unimolecular cleavage was studied using a model that only includes the nucleoside. Although comparison of experimental and (PCM-B3LYP/6-31+G(d)) calculated (Gibbs energy) barriers confirms that hydrolysis occurs via a fully dissociative (S(N)1) mechanism with a rate-limiting step of glycosidic bond dissociation, this model does not provide a complete picture of the hydrolysis mechanism. When the model is expanded to include one explicit water nucleophile, gas-phase optimizations are unable to model charge separation in the reaction intermediate, while optimizations that implicitly incorporate the effects of bulk solvent do not accurately model the second reaction step (nucleophilic attack following dissociation) due to insufficient (water) nucleophile activation and (uracil anion) leaving group stabilization. Further expansion of the model to include three explicit water molecules allows for discrete proton transfer from the water nucleophile to the uracil anion, and thereby generates smooth reaction surfaces for both the dissociative (S(N)1) and concerted (S(N)2) pathways. Furthermore, for the first time, this computational model for the uncatalyzed hydrolysis of the N-glycosidic bond in a nucleoside predicts that the dissociative mechanism is more favorable than the concerted pathway, which supports experimental findings. It is also found that although (implicit) solvent-phase single-point calculations on gas-phase geometries can yield similar energies to solvent-phase optimizations, the geometries can be very different and not all potential reaction routes can be fully characterized. Therefore, care must be taken when interpreting mechanistic information obtained from gas-phase structures. This work provides a template for generating other nucleoside or nucleotide hydrolysis models including those relevant to both uncatalyzed and enzyme-catalyzed reactions.

Human uracil-DNA glycosylase (hUNG2) is a base excision repair enzyme that removes the damaged base uracil from DNA through hydrolytic deglycosylation of the nucleotide. In the present study, the mechanism of hUNG2 is thoroughly investigated using ONIOM(MPWB1K/6-31G(d):PM3) active-site models to generate reaction potential energy surfaces. Active-site models that differ in the hydrogen-bonding arrangement of the nucleophilic water molecule and/or protonation state of His148 are considered. The large barrier calculated using the model with a cationic His148 verifies that this residue is neutral in the early stages of the reaction. The reaction pathways predicted by two models with a neutral His148 are consistent with a wealth of experimental data on the enzyme, including mutational studies, which supports our approach. On the basis of our calculations, we propose a complete mechanism for the chemical step of hUNG2. In the first part of the reaction, His268, Asn204, and a water molecule work together to stabilize the negative charge forming on the uracil moiety. Subsequently, either Asp145 or His148 can act as the general base that activates the water nucleophile depending on the binding orientation of the water molecule in the active site. However, we propose that His148 preferentially acts as the general base. Therefore, in agreement with previous proposals, we assign the primary function of Asp145 to electrostatic stabilization of the positive charge developing on the sugar moiety during the reaction, which is also consistent with a growing theory that the primary function of active-site carboxylate groups present in many glycosylases is transition state stabilization. Most importantly, our work explains, for the first time, the role of His148 in the chemical step and provides additional support for the inclusion of this amino acid in the list of residues (Asp145 and His268) essential to the chemical step of the hUNG2 mechanism.

Deglycosylation of nucleotides occurs during many essential biological processes, including DNA repair, and is initiated by a variety of nucleophiles. In the present work, density functional theory (B3LYP) was used to investigate the thermodynamics and kinetics of the glycosidic bond cleavage reaction in the model nucleoside forms of guanine and its major oxidation product, 8-oxoguanine. Base excision facilitated by four different nucleophiles (hydroxyl anion (fully activated water), formate-water complex (partially activated water), lysine, and proline) was considered, which spans nucleophiles involved in a collection of spontaneous and enzyme-catalyzed processes. Because some enzymes that catalyze deglycosylation can accommodate more than one orientation of the base with respect to the sugar moiety, the effects of the (anti/syn) base orientation on the barrier height were also considered. We find that the nucleophile has a very large effect on the overall (gas-phase) reaction energetics. Although this effect decreases in different (polar) environments, the nucleophile has the greatest influence on the overall reaction as compared to whether the base is damaged or to the base orientation. Furthermore, the effects are significant in environments that most closely resemble (nonpolar) enzymatic active sites. Our results provide a greater understanding of the relative effects of the nucleophile, damage to the nucleobase, and the nucleobase orientation with respect to the sugar moiety on the deglycosylation pathway, which provide qualitative explanations for relative base excision rates observed in some biological systems.

Two-dimensional PCM-B3LYP/6-31+G(d) potential energy surfaces for the hydrolysis of the four natural 2'-deoxyribonucleosides (2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine, and thymidine) are characterized using a model that includes both implicit (bulk) solvent effects and (three or four) explicit water molecules in the optimization routine. For the first time, the experimentally predicted dissociative (S(N)1) mechanism is found to be favored over the synchronous (S(N)2) pathway for all nucleosides studied. Due to the success of our model in stabilizing the charge-separated intermediates along the S(N)1 pathway, it is proposed that the new model presented here is the smallest system capable of generating the experimentally predicted oxacarbenium cation intermediate. We therefore stress that dissociative mechanisms should be studied with methodologies that account for the (bulk) environment in the optimization routine, where these effects are often only included as a correction to the energy in the current literature. In addition to accounting for charge stabilization through implicit solvation, nucleophile activation and leaving group stabilization should also be explicitly introduced into the model to further stabilize the system. Our work also emphasizes the importance of studying the Gibbs surface, which in some cases provides a better description of chemically important regions of the reaction surface or changes the calculated trend in the magnitude of dissociative barriers. In addition, it is proposed that the methodology presented in this study can be used to calculate uncatalyzed deglycosylation barriers for a range of DNA nucleosides, which when compared to the corresponding enzyme-catalyzed reactions, will allow the prediction of the rate enhancement (barrier reduction) due to the enzyme.