Preorganized Internal Electric Field Powers Catalysis in the Active Site of Uracil-DNA Glycosylase

Abstract

Uracil-DNA glycosylase (UDG) is a monofunctional DNA glycosylase, which is involved in the base excision repair (BER) pathway and responsible for the excision of uracil from DNA. UDG is well known for its high catalytic efficiency and substrate autocatalysis character. Here, using quantum-mechanical/molecular-mechanical (QM/MM) and QM calculations as well as molecular dynamics (MD) simulations, we propose a revised catalytic mechanism of UDG and elucidate the nature of its strong catalytic efficiency. In the initial stage of the reaction, a heterolytic C−N bond cleavage is catalyzed by a strong internal electric field which stabilizes the charge distribution of the transition state more than that of the ground state, yielding an oxocarbenium-ion−uracil-anion species. The catalytic effect of substrate phosphate groups can be included in the effect of the internal electric field, which is counterbalanced by sodium ions, explaining the longstanding discrepancy between theory and experiment. Subsequently, Asp145 acts as the general base to activate the water nucleophile to attack the oxocarbenium ion, forming an abasic site. Interestingly, the proton further transfers from Asp145 to the neutral and more solvent-exposed residue His148 to facilitate its release, which is demonstrated to be driven by the internal electric field. This work deepens our understanding of the catalytic mechanism of UDG and more generally the catalytic effect of internal electric fields in enzymes.

How the Conformational Movement of the Substrate Drives the Regioselective C–N Bond Formation in P450 TleB: Insights from Molecular Dynamics Simulations and Quantum Mechanical/Molecular Mechanical Calculations

“…The IEF was computed as described in previous studies. , Briefly, it is calculated with Coulomb’s law, whereby the atoms are treated as point charges and their positions are determined by the QM/MM-optimized structures. The magnitude of the point charge was obtained from the AMBER ff14SB force field and TIP3P model for the protein residues and water molecules, respectively.…”

Preorganized Internal Electric Field Promotes a Double-Displacement Mechanism for the Adenine Excision Reaction by Adenine DNA Glycosylase

“…3.1 Å above the anomeric carbon in a representative snapshot of the MD simulation trajectory, is ready for the nucleophilic attack. Intriguingly, we find that the formation of the C–O bond precedes significantly the removal of the proton from the nucleophilic water in MutY, which is in contrast with the case of UDG where they occur almost simultaneously. ,,, As shown in Figure , the attack substep (Int2′ → Int3) has a moderate barrier of 11.2 kcal/mol (TS3 in Figure b; refer to Figure S15 for the QM/MM-obtained TS3 structure). Without activation the Int3 intermediate lies 3.2 kcal/mol higher than the Int2′ species.…”

“…An intriguing question is how the enzyme achieves this function. In view of the crucial role of the IEF found previously in our previous study of UDG, we calculated the IEF at the position of the anomeric carbon in MutY. Interestingly, the IEF was found to point to the 5′ side in MutY (refer to Figure ), which is opposite to the direction of the IEF found in UDG .…”

Evaluating the Transition State Stabilization/Destabilization Effects of the Electric Fields from Scaffold Residues by a QM/MM Approach

“…Theoretical studies also provide significant insights into the catalytic effects of electric fields in enzymes. For instance, Warshel et al employed the empirical valence bond (EVB) method to quantify the catalytic effect of the preorganized electrostatic environment in various enzymes. ,,− Alexandrova’s group have shown that the effect of electrostatic preorganization can be reflected by the change of geometry of charge density in the theoretical framework of Quantum Theory of Atoms in Molecules (QTAIM). − Head-Gordon’s group evaluated the transition-state free energy stabilization based on the interaction energy of residue electric fields with bond dipoles in the reactant and TSs, which successfully guide the optimization of the electric field for improving the catalytic efficiency of a designed Kemp eliminase through mutation in the protein scaffold residues. ,, The application of oriented external electric fields (OEEFs) in computational models has also been used to infer how the electric fields affect the energetics of chemical reactions. ,,− …”

“…In addition to its active site, an enzyme includes a protein scaffold that consists of the majority of enzyme residues. The protein scaffold not only plays key roles in the substrate/cofactor binding and substance transportation − but also is vital to the boost of catalytic efficiency of the enzyme via the electrostatic stabilization effect. − Especially, the scaffold residues are shown to contribute to the preorganized electric field in the active site of natural enzymes, which can stabilize the charge distribution of the transition state (TS) more than that of the reactant state (RS), leading to acceleration of the catalytic rate. ,− In contrast to natural enzymes that can optimize their protein scaffold through billions of years of evolution, the design of de novo enzymes mainly focuses on the active site, while the scaffolds of the de novo enzymes may lack sufficient electrostatic preorganization as in natural enzymes, which limits the catalytic efficiency of the de novo enzymes. , Accordingly, systematic evaluation of the TS stabilization (or destabilization) effects of the electric fields generated by the scaffold residues is key to both understanding the emergence of the eminent catalytic activity of natural enzymes and the rational design of de novo enzymes.…”