Role of active site residues in the glycosylase step of T4 Endonuclease V.Computer simulation studies on ionization states

T4 Endonuclease V (EndoV) is a base excision repair enzyme that removes thy mine dimers (TD) from damaged DNA. To elucidate the role of the active site residues in catalysis, their pK(a)'s were evaluated using the semimicrosco pic version of the protein dipoles-langevin dipoles method (PDLD/S). Contri butions of different effects to the pK(a) such as charge-charge interaction s, conformational rearrangement, protein relaxation, and DNA binding were a nalyzed in detail. Charging of the active site residues was found to be les s favorable in the complex than in the free enzyme. The pK(a) of the N-term inus decreased from 8.01 in the free enzyme to 6.52 in the complex, while t he pK(a) of Glu-23 increased from 1.52 to 7.82, which indicates that the ke y residues are neutral in the reactant state of the glycosylase step. These pK(a)'s are in agreement with the optimal pH range of the reaction and sup port the N-terminus acting as a nucleophile. The Glu-23 in its protonated f orm 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 nucleoph ilic 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 electros tatically favorable environment for the leaving base. To distinguish betwee n S(N)1 and S(N)2 mechanisms of the glycosylase step the energetics of prot onating O2 of 5' TD was calculated. The enzyme was found to stabilize the n eutral thymine by similar to 3.6 kcal/mol, whereas it destabilizes the prot onated thymine by similar to 6.6 kcal/mol with respect to an aqueous enviro nment. Consequently, the formation of a protonated thymine intermediate is unlikely, indicating an S(N)2 reaction mechanism for the glycosylase step.