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 ill bacteria by removing oxidized guanine bases from DNA and can also cleave the nascent or preformed abasic DNA by P,beta,delta-elimination. In this work, we have used the quench-flow technique (1) 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-now 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.