Catalysis by members of the RNase H superfamily of enzymes is generally believed
to require only two Mg2+ ions that are coordinated by active-site
carboxylates. By examining the catalytic process of Bacillus halodurans RNase H1
in crystallo, however, we found that the two canonical Mg2+ ions and
an additional K+ failed to align the nucleophilic water for RNA
cleavage. Substrate alignment and product formation required a second
K+ and a third Mg2+, which replaced the first
K+ and departed immediately after cleavage. A third transient
Mg2+ has also been observed for DNA synthesis, but in that case it
coordinates the leaving group instead of the nucleophile as in the case of the
RNase H1 hydrolysis reaction. These transient cations have no contact with the
enzymes. Other DNA and RNA enzymes that catalyze consecutive cleavage and
strand-transfer reactions in a single active site may similarly require cation
trafficking coordinated by the substrate.
