title: The acidic triad conserved in type IA DNA topoisomerases is required for binding of Mg(II) and subsequent conformational change.
abstract: The acidic residues Asp-111, Asp-113, and Glu-115 of Escherichia coli DNA topoisomerase I are located near the active site Tyr-319 and are conserved in type IA topoisomerase sequences with counterparts in type IIA DNA topoisomerases. Their exact functional roles in catalysis have not been clearly defined. Mutant enzymes with two or more of these residues converted to alanines were found to have >90% loss of activity in the relaxation assay with 6 mM Mg(II) present. Mg(II) concentrations (15-20 mM) inhibitory for the wild type enzyme are needed by these double mutants for maximal relaxation activity. The triple mutant D111A/D113A/E115A had no detectable relaxation activity. Mg(II) binding to wild type enzyme resulted in an altered conformation detectable by Glu-C proteolytic digestion. This conformational change was not observed for the triple mutant or for the double mutant D111A/D113A. Direct measurement of Mg(II) bound showed the loss of 1-2 Mg(II) ions for each enzyme molecule due to the mutations. These results demonstrate a functional role for these acidic residues in the binding of Mg(II) to induce the conformational change required for the relaxation of supercoiled DNA by the enzyme.
authors.affiliation: Department of Biochemistry, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: C X
authors.initials: CX
authors.lastname: Zhu
authors.forename: Y C
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D017124:Conserved Sequence; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000201:enzymology; D008274:Magnesium / Q000378:metabolism*; D015636:Magnesium Chloride / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
chemical_list: D015636:Magnesium Chloride; D004264:DNA Topoisomerases, Type I; D008274:Magnesium; D000409:Alanine