PMID:19835417
| Citation |
Kuznetsov, NA, Zharkov, DO, Koval, VV, Buckle, M and Fedorova, OS (2009) Reversible chemical step and rate-limiting enzyme regeneration in the reaction catalyzed by formamidopyrimidine-DNA glycosylase. Biochemistry 48:11335-43 |
|---|---|
| Abstract |
Formamidopyrimidine-DNA N-glycosylase (Fpg) operates in the base excision repair pathway in bacteria by removing oxidized guanine bases from DNA and can also cleave the nascent or preformed abasic DNA by beta,delta-elimination. In this work, we have used the quench-flow technique (i) to show that the kinetics of processing of 7,8-dihydro-8-oxoguanine and abasic site lesions by Fpg from Escherichia coli involves a burst phase and a stationary phase, (ii) to establish the reaction kinetic scheme, and (iii) to calculate the rate constants for the reaction steps. A comparison of the quench-flow results with the data from earlier stopped-flow kinetics with tryptophan and 2-aminopurine fluorescence detection reveals that the cleaved product formation is initially reversible; it is followed by conformational changes in the enzyme and DNA molecules that represent the postchemical irreversible rate-limiting steps. We have applied mass spectrometry with electrospray ionization to follow the appearance and disappearance of transient covalent intermediates between Fpg and the substrate DNA. The overall rate-limiting step of the enzymatic reaction seems to be the release of Fpg from its adduct with the 4-oxo-2-pentenal remnant of the deoxyribose moiety formed as a result of DNA strand cleavage by beta,delta-elmination. |
| Links |
PubMed Online version:10.1021/bi901100b |
| Keywords |
Algorithms; Base Sequence; Binding Sites; Biocatalysis; DNA/chemistry; DNA/metabolism; DNA-Formamidopyrimidine Glycosylase/chemistry; DNA-Formamidopyrimidine Glycosylase/metabolism; Escherichia coli/enzymology; Guanine/analogs & derivatives; Guanine/chemistry; Guanine/metabolism; Kinetics; Protein Conformation; Spectrometry, Fluorescence; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity |
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