Reversible Chemical Step and Rate-Limiting Enzyme Regeneration in the Reaction Catalyzed by Formamidopyrimidine-DNA Glycosylase

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• 作者：Nikita A. Kuznetsov ; Dmitry O. Zharkov ; Vladimir V. Koval ; Malcolm Buckle ; Olga S. Fedorova
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：December 8, 2009
• 年：2009
• 卷：48
• 期：48
• 页码：11335-11343
• 全文大小：1025K
• 年卷期：v.48,no.48(December 8, 2009)
• ISSN：1520-4995

文摘

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 β,δ-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 β,δ-elmination.