Dispensability of glutamic acid 48 and aspartic acid 134 for Mn2+-dependent activity of Escherichia coli ribonuclease HI

The activities of the eight mutant proteins of Escherichia coli RNase HI, in which the four carboxylic amino acids (Asp(10), Glu(48), Asp(70), and Asp(134)) involved in catalysis are changed to Asn (Gln) or Ala, were examined in the presence of Mn2+. Of these proteins, the E48A, E48Q, D134A, and D134N proteins exhibited the activity, indicating that Glu(48) and Asp(134) are dispensable for Mn2+-dependent activity. The maximal activities of the E48A and D134A proteins were comparable to that of the wild-type protein. However, unlike the wild-type protein, these mutant proteins exhibited the maximal activities in the presence of > 100 muM MnCl2, and their activities were not inhibited at higher Mn2+ concentrations (up to 10 mM). The wild-type protein contains two Mn2+ binding sites and is activated upon binding of one Mn2+ ion at site 1 at low (similar to1 muM) Mn2+ concentrations. This activity is attenuated upon binding of a second Mn2+ ion at site 2 at high (> 10 muM) Mn2+ concentrations. The cleavage specificities of the mutant proteins, which were examined using oligomeric substrates at high Mn2+ concentrations, were identical to that of the wild-type protein at low Mn2+ concentrations but were different from that of the wild-type protein at high Mn2+ concentrations. These results suggest that one Mn2+ ion binds to the E48A, E48Q, D134A, and D134N proteins at site 1 or a nearby site with weaker affinities. The binding analyses of the Mn2+ ion to these proteins in the absence of the substrate support this hypothesis. When Mn2+ ion is used as a metal cofactor, the Mn2+ ion itself, instead of Glu(48) and Asp(134), probably holds water molecules required for activity.