Significant kinetic solvent isotope effects in folding of the catalytic RNA from the hepatitis delta virus.

The exchange of deuterium for hydrogen in water often produces solvent kinetic isotope effects (KSIEs) on the rate constants associated with enzyme reactions, including those catalyzed by RNA. Recently, KSIEs have been used to show that proton transfer occurs in the rate-limiting step of cleavage by the hepatitis delta virus (HDV) ribozyme and other catalytic RNAs. To test the underlying assumption that KSIEs are related to the chemistry step of ribozyme-mediated cleavage reactions, we developed fluorescence resonance energy transfer assays to measure KSIEs on the rate constants of conformational changes associated with substrate binding and dissociation by a trans-acting HDV ribozyme. We observe comparable KSIEs ( approximately 2-2.5-fold) of rate constants of conformational change and cleavage, while proton inventory experiments are consistent with a shift in the ensemble of transition states upon increase of D2O in the solvent. Taken together, these results challenge the common assumption that pL profiles of RNA-catalyzed reactions yielding a pKa and KSIE necessarily provide evidence for an ionization (chemistry) step to be rate-limiting. They also suggest that an unusual proton inventory may provide a signature for a conformational change contributing to the rate-limiting step.