title: Lys98 substitution in human AP endonuclease 1 affects the kinetic mechanism of enzyme action in base excision and nucleotide incision repair pathways.
abstract: Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme in the base excision repair (BER) and nucleotide incision repair (NIR) pathways. We recently analyzed the conformational dynamics and kinetic mechanism of wild-type (wt) protein, in a stopped-flow fluorescence study. In this study, we investigated the mutant enzyme APE1K98A using the same approach. Lys98 was known to hydrogen bond to the carboxyl group of Asp70, a residue implicated in binding the divalent metal ion. Our data suggested that the conformational selection and induced fit occur during the enzyme action. We expanded upon the evidence that APE1 can pre-exist in two conformations. The isomerization of an enzyme-product complex in the BER process and the additional isomerization stage of enzyme-substrate complex in the NIR process were established for APE1K98A. These stages had not been registered for the wtAPE1. We found that the K98A substitution resulted in a 12-fold reduction of catalytic constant of 5'-phosphodiester bond hydrolysis in (3-hydroxytetrahydrofuran-2-yl)methyl phosphate (F, tetrahydrofuran) containing substrate, and in 200-fold reduction in 5,6-dihydrouridine (DHU) containing substrate. Thus, the K98A substitution influenced NIR more than BER. We demonstrated that the K98A mutation influenced the formation of primary unspecific enzyme-substrate complex in a complicated manner, depending on the Mg(2+) concentration and pH. This mutation obstructed the induced fit of enzyme in the complex with undamaged DNA and F-containing DNA and appreciably decreased the stability of primary complex upon interaction of enzyme with DNA, containing the natural apurinic/apyrimidinic (AP) site. Furthermore, it significantly delayed the activation of the less active form of enzyme during NIR and slowed down the conformational conversion of the complex of enzyme with the cleavage product of DHU-substrate. Our data revealed that APE1 uses the same active site to catalyze the cleavage of DHU- and AP-substrates.
authors.affiliation: Siberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia.
authors.forename: Nadezhda A
authors.initials: NA
authors.lastname: Timofeyeva
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Koval
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishchenko
authors.forename: Murat K
authors.initials: MK
authors.lastname: Saparbaev
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D019943:Amino Acid Substitution; D001483:Base Sequence; D002021:Buffers; D020134:Catalytic Domain; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008024:Ligands; D008239:Lysine; D009154:Mutation; D013050:Spectrometry, Fluorescence; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D002021:Buffers; D008024:Ligands; C010922:5,6-dihydrouridine; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D008239:Lysine; D014529:Uridine