title: The role of Asn-212 in the catalytic mechanism of human endonuclease APE1: stopped-flow kinetic study of incision activity on a natural AP site and a tetrahydrofuran analogue.
abstract: Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ∼550-fold, while Ala substitution results in ∼70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding.
authors.affiliation: Institute of Chemical Biology & Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk 630090, Russia; Novosibirsk State University, Pirogova St., 2, Novosibirsk 630090, Russia.
authors.forename: Lyubov Yu
authors.initials: LY
authors.lastname: Kanazhevskaya
authors.affiliation: Institute of Chemical Biology & Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk 630090, Russia; Novosibirsk State University, Pirogova St., 2, Novosibirsk 630090, Russia.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Koval
authors.affiliation: Institute of Chemical Biology & Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk 630090, Russia; Novosibirsk State University, Pirogova St., 2, Novosibirsk 630090, Russia.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Lomzov
authors.affiliation: Institute of Chemical Biology & Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Lavrentyev Ave., 8, Novosibirsk 630090, Russia; Novosibirsk State University, Pirogova St., 2, Novosibirsk 630090, Russia. Electronic address: fedorova@niboch.nsc.ru.
authors.forename: Olga S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005663:Furans / Q000737:chemistry; D006801:Humans; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D005663:Furans; C018674:tetrahydrofuran; D001216:Asparagine; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase
keywords: 2-Aminopurine; APE1; Abasic site; Base excision repair; MD simulations; Pre-steady-state kinetics; Stopped-flow method