Journal
BIOCHEMISTRY
Volume 52, Issue 37, Pages 6499-6514Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi4000673
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Funding
- National Institutes of Health (NIH) [R01GM095923, GM056207]
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Metal ion and nucleobase catalysis are important for ribozyme mechanism, but the extent to which they cooperate is unclear. A crystal structure of the hepatitis delta virus (HDV) ribozyme suggested that the pro-Re oxygen at the scissile phosphate directly coordinates a catalytic Mg2+ ion and is within hydrogen bonding distance of the amine of the general acid C75. Prior studies of the genomic HDV ribozyme, however, showed neither a thio effect nor metal ion rescue using Mn2+. Here, we combine experiment and theory to explore phosphorothioate substitutions at the scissile phosphate. We report significant thio effects at the scissile phosphate and metal ion rescue with Cd2+. Reaction profiles with an Sp-phosphorothioate substitution are indistinguishable from those of the unmodified substrate in the presence of Mg2+ or Cd2+, supporting the idea that the pro-S-p, oxygen does not coordinate metal ions. The Rp-phosphorothioate substitution, however, exhibits biphasic kinetics, with the fast-reacting phase displaying a thio effect of up to 5-fold and the slow-reacting phase displaying a thio effect of similar to 1000-fold. Moreover, the fast- and slow-reacting phases give metal ion rescues in Cd2+ of up to 10- and 330-fold, respectively. The metal ion rescues are unconventional in that they arise from Cd2+ inhibiting the oxo substrate but not the R-p substrate. This metal ion rescue suggests a direct interaction of the catalytic metal ion with the pro-Re oxygen, in line with experiments with the antigenomic HDV ribozyme. Experiments without divalent ions, with a double mutant that interferes with Mg2+ binding, or with C75 deleted suggest that the pro-Re oxygen plays at most a redundant role in positioning C75. Quantum mechanical/molecular mechanical (QM/MM) studies indicate that the metal ion contributes to catalysis by interacting with both the pro-R-p oxygen and the nucleophilic 2'-hydroxyl, supporting the experimental findings.
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