title: Mutation of a unique aspartate residue abolishes the catalytic activity but not substrate binding of the mouse N-methylpurine-DNA glycosylase (MPG).
abstract: N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a variety of alkylated purine adducts. Although Asp was identified as the active site residue in various DNA glycosylases based on the crystal structure, Glu-125 in human MPG (Glu-145 in mouse MPG) was recently proposed to be the catalytic residue. Mutational analysis for all Asp residues in a truncated, fully active MPG protein showed that only Asp-152 (Asp-132 in the human protein), which is located near the active site, is essential for catalytic activity. However, the substrate binding was not affected in the inactive Glu-152, Asn-152, and Ala-152 mutants. Furthermore, mutation of Asp-152 did not significantly affect the intrinsic tryptophan fluorescence of the enzyme and the far UV CD spectra, although a small change in the near UV CD spectra of the mutants suggests localized conformational change in the aromatic residues. We propose that in addition to Glu-145 in mouse MPG, which functions as the activator of a water molecule for nucleophilic attack, Asp-152 plays an essential role either by donating a proton to the substrate base and, thus, facilitating its release or by stabilizing the steric configuration of the active site pocket.
authors.affiliation: Sealy Center for Molecular Science and Department of Human Biological Chemistry and Genetics, University of Texas, Medical Branch, Galveston, Texas 77555-1079, USA. raroy@utmb.edu
authors.forename: R
authors.initials: R
authors.lastname: Roy
authors.forename: T
authors.initials: T
authors.lastname: Biswas
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
authors.forename: S
authors.initials: S
authors.lastname: Mitra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D001665:Binding Sites; D002942:Circular Dichroism; D018983:DNA Footprinting; D045647:DNA Glycosylases; D004260:DNA Repair; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence
chemical_list: D011994:Recombinant Proteins; D001224:Aspartic Acid; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II