ROLE OF THE MG2-COLI RIBONUCLEASE HI REACTION( ION IN THE ESCHERICHIA)

To study the interaction and the role of the metal ion in the reaction catalyzed by Escherichia coli ribonuclease HI (E. coli RNase HI), sub strate analogues containing a phosphorothioate linkage or 2'-modified nucleosides at the cleavage site were used, In the presence of Mg2+, M n2+, Co2+, Zn2+, or Cd2+, the phosphorothioate linkage with the R(P)-c onfiguration was cleaved, while the S-P-isomer was not. Kinetic studie s showed that Mn2+ and Cd2+ facilitated the cleavage of the phosphorot hioate to only a small extent, which indicated the absence of an inter action between the metal ion and this phosphate residue. The interacti on of the metal ion with the 2'-functional group was analyzed by Mg2+- titration experiments using the -OH, -NH2, and -F substrates. From Hil l plots, it was found that the K-Mg values were almost the same. These results are evidence of an interaction between Mg2+ and the 2'-functi onal group by the formation of an outer-sphere complex with a water mo lecule. The Hill coefficient of 1.0 for the -OH substrate indicated th at a single Mg2+ ion is required for the catalysis.