Reversible chemical step and rate-limiting enzyme regeneration in the reaction catalyzed by formamidopyrimidine-DNA glycosylase.
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.
Kuznetsov
Nikita A
NA
Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia.
Zharkov
Dmitry O
DO
Koval
Vladimir V
VV
Buckle
Malcolm
M
Fedorova
Olga S
OS
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites; D055162:Biocatalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
C453560:7,8-dihydro-8-oxoguanine; D006147:Guanine; D004247:DNA; D043382:DNA-Formamidopyrimidine Glycosylase