The catalytic mechanism of Fpg protein. Evidence for a Schiff base intermediate and amino terminus localization of the catalytic site.
Our recent structure-activity analysis of Fpg protein of Escherichia coli, using oligodeoxynucleotides containing various 8-oxopurine derivatives, has allowed us to postulate an enzyme mechanism involving protonation of 8-oxoguanine at O-6 and nucleophilic attack of the deoxyribose moiety at C-1' leading to the formation of an enzyme-substrate Schiff base intermediate (Tchou, J., Bodepudi, V., Shibutani, S., Antoshechkin, I., Miller, J., Grollman, A. P., and Johnson, F. (1994) J. Biol. Chem. 269, 15318-15324). In this paper, sodium cyanoborohydride has been used to convert the transient intermediate to a covalent enzyme-DNA complex. The location of the active site of Fpg protein is further delineated using two approaches. 1) A radiolabeled DNA substrate is used to tag the active site of Fpg protein, using sodium cyanoborohydride. The active site is mapped to the first 73 amino acid residue fragment by cyanogen bromide cleavage analysis. 2) A maltose-binding protein fusion system is used to generate amino-terminal modifications of Fpg protein to explore the role of the amino-terminal region in DNA binding and catalysis. Results support the conclusion that the active site of Fpg protein is located at or near the amino terminus. Thus, Fpg protein may act in a similar fashion as T4 endonuclease V, a DNA repair enzyme that uses its amino-terminal alpha-amino group of threonine to carry out catalysis via Schiff base formation (Dodson et al., 1993).
Tchou
J
J
Department of Pharmacological Sciences, State University of New York, Stony Brook 11794-8651, USA.
Grollman
A P
AP
D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism; D043382:DNA-Formamidopyrimidine Glycosylase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism; D005798:Genes, Bacterial; D005838:Genotype; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012545:Schiff Bases
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012545:Schiff Bases; C461599:maltose transport system, E coli; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D015951:Factor Xa