Mechanism of action of Escherichia coli formamidopyrimidine N-glycosylase: role of K155 in substrate binding and product release.
Escherichia coli formamidopyrimidine N-glycosylase (fpg) is a DNA glycosylase with an associated beta,delta-lyase activity. We have recently shown that the highly conserved lysine residue K155 is important for base recognition. Incubation of a double-stranded DNA containing an abasic site with the wild-type fpg protein generated only beta,delta-product. However, incubation of a double-stranded DNA containing an abasic site opposite a small gap with fpg protein generated predominantly beta-product. These data suggested that the induction of a double-strand break by fpg led to the destabilization of the protein-DNA covalent intermediate, causing the fpg protein to prematurely dissociate from the DNA substrate. Furthermore, when a double-stranded DNA containing an abasic site opposite an A was used as a substrate, K155A mutant fpg protein yielded a mixture of beta- and beta,delta-products. These data suggested that K155 is essential for maintaining the stability of the intermediary protein-DNA covalent complex. Pre-steady-state burst kinetics showed that mutation in K155 led to the apparent disappearance of the initial burst, suggesting that the rate of product release from K155A is much greater than the rate of chemical reaction catalyzed by the mutant enzyme. This is consistent with the idea that K155A dissociates prematurely from the covalent complex, leading to a higher turnover number observed for K155A for DNA substrate containing an AP site.
Rabow
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Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, Atlanta, Georgia 30335, USA.
Venkataraman
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R
Kow
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YW
D001080:Apurinic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D019757:Carbon-Oxygen Lyases / Q000378:metabolism; D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D043382:DNA-Formamidopyrimidine Glycosylase; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D001080:Apurinic Acid; D004247:DNA; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase