Implications of Alternative Substrate Binding Modes for Catalysis by Uracil-​DNA Glycosylase: An Apparent Discrepancy Resolved

A theor. study showed that the base excision repair enzyme uracil-​DNA glycosylase (UDG) exploits electrostatic interactions with backbone phosphate groups in the substrate for catalysis. Although expts. performed to test the calcd. results confirmed the predicted importance of the -​2, -​1, and +1 phosphate groups, there was an apparent disagreement with regard to the +2 phosphate group. The calcns. indicated that it made an important contribution, while exptl., the effect of its deletion or neutralization was small. The +2 phosphate group interacts directly with an active site histidine (H148 in humans) in the crystal structure of UDG in complex with double-​stranded (ds) DNA. We previously calcd. that H148 has a strong anticatalytic effect due to its protonation, and here we use alchem. free energy simulations to est. its site-​specific pKa. We find that it is pos. charged over the entire exptl. pH range (4-​10)​, so its deprotonation cannot compensate for deletion or neutralization of the +2 phosphate group. The free energy simulations are facilitated by an efficient charge-​scaling procedure that allows quant. correction for the implicit treatment of solvent far from the active site; improvements are made to that method to account carefully for differences in the truncation of electrostatic interactions in the contributing mol.-​mech. and continuum-​electrostatic approaches. Addnl. simulations are used to demonstrate that the +2 phosphate group is fully solvent exposed in complexes with single-​stranded DNA substrates like those used in the expts. In contrast, it is well-​structured and buried in the dsDNA complex used in the original simulations. Differences in solvent shielding thus account for the apparent lack of an effect obsd. exptl. upon neutralization or deletion of this group.