Mechanism of Action of Escherichia coli Ribonuclease III. Stringent Chemical Requirement for the Glutamic Acid 117 Side Chain and Mn2+ Rescue of the Glu117Asp Mutant

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Abstract
Escherichia coli ribonuclease III (EC 3.1.24) is a double-strand- (ds-) specific endoribonuclease involved in the maturation and decay of cellular, phage, and plasmid RNAs. RNase III is a homodimer and requires Mg2+ to hydrolyze phosphodiesters. The RNase III polypeptide contains an N-terminal catalytic (nuclease) domain which exhibits eight highly conserved acidic residues, at least one of which (Glu117) is important for phosphodiester hydrolysis but not for substrate binding [Li and Nicholson (1996) EMBO J. 15, 1421−1433]. To determine the side chain requirements for activity, Glu117 was changed to glutamine or aspartic acid. The mutant proteins were purified as (His)6-tagged species, and both exhibited normal homodimeric behavior as shown by chemical cross-linking. The Glu117Gln mutant is unable to cleave substrate in vitro under all tested conditions but can bind substrate. The Glu117Asp mutant also is defective in cleavage while able to bind substrate. However, low level activity is observed at extended reaction times and high enzyme concentrations, with an estimated catalytic efficiency ∼15 000-fold lower than that of RNase III. The activity of the Glu117Asp mutant but not that of the Glu117Gln mutant can be greatly enhanced by substituting Mn2+ for Mg2+, with the catalytic efficiency of the Glu117Asp−Mn2+ holoenzyme ∼400-fold lower than that of the RNase III−Mn2+ holoenzyme. For RNase III, a Mn2+ concentration of 1 mM provides optimal activity, while concentrations >5 mM are inhibitory. In contrast, the Glu117Asp mutant is not inhibited by high concentrations of Mn2+. Finally, high concentrations of Mg2+ do not inhibit RNase III nor relieve the Mn2+-dependent inhibition. In summary, these experiments establish the stringent functional requirement for a precisely positioned carboxylic acid group at position 117 and reveal two classes of divalent metal ion binding sites on RNase III. One site binds either Mg2+ or Mn2+ and supports catalysis, while the other site is specific for Mn2+ and confers inhibition. Glu117 is important for the function of both sites. The implications of these findings on the RNase III catalytic mechanism are discussed.