Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease.

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5' side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3'-5'-exonuclease, 3'-phosphodiesterase, and 3'-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre-steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding-site residue, Arg181, was analyzed too; it changed its conformation when enzyme-substrate and enzyme-product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction.