The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

N. A. Kuznetsov, A. S. Kiryutin, A. A. Kuznetsova, M. S. Panov, M. O. Barsukova, A. V. Yurkovskaya, O. S. Fedorova*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)950-967
Number of pages18
JournalJournal of Biomolecular Structure and Dynamics
Issue number5
StatePublished - Apr 4 2017

Bibliographical note

Funding Information:
This work was supported by Russian Ministry of Education and Science [SS-7564.2016.4]; the Russian Academy of Sciences, “Molecular & Cell Biology” Program [grant number 6.11]; Russian Foundation for Basic Research [14-03-00453], [14-04-00531], [15-04-00467], [15-34-20121], [16-04-00037]. Part of the work with stopped-flow experiments was specifically funded by Russian Science Foundation [grant number. 14-14-00063]. NMR data collection and analysis were separately funded by Russian Science Foundation [grant number. 15-13-20035].

Publisher Copyright:
© 2016 Informa UK Limited, trading as Taylor & Francis Group.


  • alkyladenine DNA glycosylase
  • base excision repair
  • conformational dynamics
  • DNA glycosylases
  • enzyme kinetics
  • NMR

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


Dive into the research topics of 'The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase'. Together they form a unique fingerprint.

Cite this