Direct pKa Measurement of the Active-Site Cytosine in a Genomic Hepatitis Delta Virus Ribozyme

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Abstract
Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pKa consistent with its role as a general acid or base, we used 13C NMR to determine its microscopic pKa in the product form of the ribozyme. The measured pKa is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pKa's measured kinetically. However, under all conditions tested, the microscopic pKa is lower than the apparent reaction pKa, supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pKa is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pKa in an intermediate state of the transesterification reaction.