Much is known about mechanistic strategies for protein enzymes, but less is understood for RNA enzymes. The hepatitis delta virus (HDV) RNA enzyme is a small self-cleaving RNA that is widespread in biology. The ribozyme carries out cleavage of a single phosphodiester bond with exceptional rate enhancement. We conducted a wealth of kinetics experiments on this ribozyme, including solvent isotope effects, proton inventories, thio effects and metal ion rescues. In addition, we trapped the RNA in a catalytically relevant precleaved state, solved its crystal structure, and conducted Raman spectroscopic experiments on it. We additionally performed molecular dynamics (MD), electrostatic non-linear Poisson-Boltzmann (NLPB), and quantum mechanical/molecular mechanical (QM/MM) calculations on the ribozyme. Our studies lead to a unified mechanism for the ribozyme involving general acid catalysis by the protonated nucleobase C75, and Lewis acid catalysis by a catalytic magnesium ion. Electrostatics play a particularly important role in the mechanism of this ribozyme, tuning the pKa of C75, facilitating binding of the catalytic magnesium ion, and orienting the reactive atoms for catalysis. We conclude that electrostatics play a major role in catalysis of this and likely many other RNA enzymes.

Key roles for electrostatics in the mechanism of the HDV ribozyme

246th National Meeting (2013)

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