title: The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.
abstract: Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.
authors.affiliation: a Institute of Chemical Biology and Fundamental Medicine , Lavrentyev Ave. 8, Novosibirsk 630090 , Russia.|c Department of Natural Sciences , Novosibirsk State University , Pirogova St. 2, Novosibirsk 630090 , Russia.
authors.forename: N A
authors.initials: NA
authors.lastname: Kuznetsov
authors.affiliation: b International Tomography Center SB RAS, Institutskaya 3a , Novosibirsk 630090 , Russia.|c Department of Natural Sciences , Novosibirsk State University , Pirogova St. 2, Novosibirsk 630090 , Russia.
authors.forename: A S
authors.initials: AS
authors.lastname: Kiryutin
authors.affiliation: a Institute of Chemical Biology and Fundamental Medicine , Lavrentyev Ave. 8, Novosibirsk 630090 , Russia.
authors.forename: A A
authors.initials: AA
authors.lastname: Kuznetsova
authors.affiliation: b International Tomography Center SB RAS, Institutskaya 3a , Novosibirsk 630090 , Russia.|c Department of Natural Sciences , Novosibirsk State University , Pirogova St. 2, Novosibirsk 630090 , Russia.
authors.forename: M S
authors.initials: MS
authors.lastname: Panov
authors.affiliation: c Department of Natural Sciences , Novosibirsk State University , Pirogova St. 2, Novosibirsk 630090 , Russia.
authors.forename: M O
authors.initials: MO
authors.lastname: Barsukova
authors.affiliation: b International Tomography Center SB RAS, Institutskaya 3a , Novosibirsk 630090 , Russia.|c Department of Natural Sciences , Novosibirsk State University , Pirogova St. 2, Novosibirsk 630090 , Russia.
authors.forename: A V
authors.initials: AV
authors.lastname: Yurkovskaya
authors.affiliation: a Institute of Chemical Biology and Fundamental Medicine , Lavrentyev Ave. 8, Novosibirsk 630090 , Russia.
authors.forename: O S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D002384:Catalysis; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D045647:DNA Glycosylases / Q000737:chemistry* / Q000378:metabolism*; D004260:DNA Repair; D006801:Humans; D007700:Kinetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D009838:Oligodeoxyribonucleotides; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics; D044366:Transition Temperature
chemical_list: D009838:Oligodeoxyribonucleotides; D004247:DNA; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases
keywords: DNA glycosylases; NMR; alkyladenine DNA glycosylase; base excision repair; conformational dynamics; enzyme kinetics