Role of Active Site Residues in the Glycosylase Step of T4 Endonuclease V. Computer Simulation Studies on Ionization States

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Abstract
T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thymine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pKa's were evaluated using the semimicroscopic version of the protein dipoles−Langevin dipoles method (PDLD/S). Contributions of different effects to the pKa such as charge−charge interactions, conformational rearrangement, protein relaxation, and DNA binding were analyzed in detail. Charging of the active site residues was found to be less favorable in the complex than in the free enzyme. The pKa of the N-terminus decreased from 8.01 in the free enzyme to 6.52 in the complex, while the pKa of Glu-23 increased from 1.52 to 7.82, which indicates that the key residues are neutral in the reactant state of the glycosylase step. These pKa's are in agreement with the optimal pH range of the reaction and support the N-terminus acting as a nucleophile. The Glu-23 in its protonated form is hydrogen bonded to O4‘ of the sugar of 5‘ TD and can play a role in increasing the positive charge of C1‘ and, hence, accelerating the nucleophilic substitution. Furthermore, the neutral Glu-23 is a likely candidate to protonate O4‘ to induce ring opening required to complete the glycosylase step of EndoV. The positively charged Arg-22 and Arg-26 provide an electrostatically favorable environment for the leaving base. To distinguish between SN1 and SN2 mechanisms of the glycosylase step the energetics of protonating O2 of 5‘ TD was calculated. The enzyme was found to stabilize the neutral thymine by ∼3.6 kcal/mol, whereas it destabilizes the protonated thymine by ∼6.6 kcal/mol with respect to an aqueous environment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an SN2 reaction mechanism for the glycosylase step.