title: Role of the Mg2+ ion in the Escherichia coli ribonuclease HI reaction.
abstract: To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), substrate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used. In the presence of Mg2+, Mn2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the RP-configuration was cleaved, while the SP-isomer was not. Kinetic studies showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorothioate to only a small extent, which indicated the absence of an interaction between the metal ion and this phosphate residue. The interaction of the metal ion with the 2'-functional group was analyzed by Mg(2+)-titration experiments using the -OH, -NH2, and -F substrates. From Hill plots, it was found that the KMg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functional group by the formation of an outer-sphere complex with a water molecule. The Hill coefficient of 1.0 for the -OH substrate indicated that a single Mg2+ ion is required for the catalysis.
authors.affiliation: Faculty of Pharmaceutical Sciences, Science University of Tokyo.
authors.forename: Y
authors.initials: Y
authors.lastname: Uchiyama
authors.forename: S
authors.initials: S
authors.lastname: Iwai
authors.forename: Y
authors.initials: Y
authors.lastname: Ueno
authors.forename: M
authors.initials: M
authors.lastname: Ikehara
authors.forename: E
authors.initials: E
authors.lastname: Ohtsuka
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D003857:Deoxyuridine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000502:physiology*; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002413:Cations, Divalent; D007477:Ions; D008670:Metals; D009841:Oligonucleotides; D010710:Phosphates; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium; D003857:Deoxyuridine