Role of the Mg2+ ion in the Escherichia coli ribonuclease HI reaction.
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.
Uchiyama
Y
Y
Faculty of Pharmaceutical Sciences, Science University of Tokyo.
Iwai
S
S
Ueno
Y
Y
Ikehara
M
M
Ohtsuka
E
E
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*
D001426:Bacterial Proteins; D002413:Cations, Divalent; D007477:Ions; D008670:Metals; D009841:Oligonucleotides; D010710:Phosphates; D016914:Ribonuclease H; C078856:ribonuclease HI; D008274:Magnesium; D003857:Deoxyuridine