Mutation at active site lysine 212 to arginine uncouples the glycosylase activity from the lyase activity of human endonuclease III.
The human endonuclease III (hNTH1) is an important DNA glycosylase with associated abasic lyase activity. We previously demonstrated that the K212Q mutant was totally inactive, while the K212R mutant had reduced DNA glycosylase/lyase activity and could form a covalent complex with the substrate DNA upon reduction. We further characterized the biochemical properties of this K212R mutant protein. NH2- (N-) terminal sequencing in combination with mass spectrometry of the peptide-DNA adduct suggested that "opportunistic" lysine(s) in the lysine-rich N-terminal tail formed a Schiff base which might result in beta-elimination. However, simultaneous substitution of Lys-75 with Gln and deletion of first 72 residues in the N-terminal tail could not cause further alteration in the glycosylase reaction or beta-elimination event. Nonetheless, the time kinetics of K212R and its subsequent mutants showed glycosylase activity without any detectable AP-lyase activity during the first 10 min of the reaction. These results suggest that a single point mutation at the active site (K212R) uncoupled the glycosylase activity from the lyase activity. We propose that the uncoupled reaction carried out by K212R is a result of direct attack either by the nonionized form of the guanidino group of arginine which forms an unstable Schiff base that hydrolyzes prior to the beta-elimination event or by hydroxide ion to cleave the glycosylic bond. In either case this reaction is followed by a secondary beta-elimination event performed by random lysine residues primarily from the N-terminal tail region.
Liu
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Sealy Center for Molecular Science, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555, USA.
Roy
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D019943:Amino Acid Substitution; D001120:Arginine / Q000235:genetics; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D045647:DNA Glycosylases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017353:Gene Deletion; D006801:Humans; D008190:Lyases / Q000235:genetics / Q000378:metabolism*; D008239:Lysine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D001120:Arginine; D004706:Endodeoxyribonucleases; D043224:Deoxyribonuclease (Pyrimidine Dimer); C106403:NTH protein, E coli; C468347:NTHL1 protein, human; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D008190:Lyases; D008239:Lysine