Direct pK(a) measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme

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 kine tic measurements. To determine whether this cytosine (C75) in the genomic r ibozyme has an altered pK(a) consistent with its role as a general acid or base, we used C-13 NMR to determine its microscopic pK(a) in the product fo rm of the ribozyme. The measured pK(a) 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 pK(a)'s measured kinetically. Howev er, under all conditions tested, the microscopic pK(a) is lower than the ap parent reaction pK(a), supporting a model in which C75 is deprotonated in t he product form of the ribozyme at physiological pH. While additional resul ts suggest that the pK(a) is not shifted in the reactant state of the riboz yme, these data cannot rule out elevation of the C75 pK(a) in an intermedia te state of the transesterification reaction.