Research output: Contribution to journal › Article › peer-review
The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase. / Kuznetsov, N. A.; Kiryutin, A. S.; Kuznetsova, A. A. et al.
In: Journal of Biomolecular Structure and Dynamics, Vol. 35, No. 5, 04.04.2017, p. 950-967.Research output: Contribution to journal › Article › peer-review
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TY - JOUR
T1 - The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase
AU - Kuznetsov, N. A.
AU - Kiryutin, A. S.
AU - Kuznetsova, A. A.
AU - Panov, M. S.
AU - Barsukova, M. O.
AU - Yurkovskaya, A. V.
AU - Fedorova, O. S.
N1 - Publisher Copyright: © 2016 Informa UK Limited, trading as Taylor & Francis Group.
PY - 2017/4/4
Y1 - 2017/4/4
N2 - 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.
AB - 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.
KW - alkyladenine DNA glycosylase
KW - base excision repair
KW - conformational dynamics
KW - DNA glycosylases
KW - enzyme kinetics
KW - NMR
KW - Oligodeoxyribonucleotides
KW - Humans
KW - Substrate Specificity
KW - DNA Glycosylases/chemistry
KW - Thermodynamics
KW - DNA/chemistry
KW - DNA Repair
KW - Nuclear Magnetic Resonance, Biomolecular
KW - Protein Binding
KW - Catalysis
KW - Kinetics
KW - Transition Temperature
KW - STEADY-STATE KINETICS
KW - ESCHERICHIA-COLI
KW - SACCHAROMYCES-CEREVISIAE
KW - CONFORMATIONAL DYNAMICS
KW - NUCLEIC-ACIDS
KW - HUMAN 8-OXOGUANINE-DNA GLYCOSYLASE
KW - ALKYLATING-AGENTS
KW - BASE-EXCISION
KW - N-GLYCOSYLASE
KW - HYDROGEN-EXCHANGE
UR - http://www.scopus.com/inward/record.url?scp=84963864305&partnerID=8YFLogxK
UR - https://www.mendeley.com/catalogue/65f38e6b-1aa4-3f3b-9f84-0e0675f82029/
U2 - 10.1080/07391102.2016.1171800
DO - 10.1080/07391102.2016.1171800
M3 - Article
C2 - 27025273
AN - SCOPUS:84963864305
VL - 35
SP - 950
EP - 967
JO - Journal of Biomolecular Structure and Dynamics
JF - Journal of Biomolecular Structure and Dynamics
SN - 0739-1102
IS - 5
ER -
ID: 8681320