Involvement of a cytosine side chain in proton transfer in the rate-determining step of ribozyme self-cleavage

Ribozymes of hepatitis delta virus have been proposed to use an active-site cytosine as an acid-base catalyst in the self-cleavage reaction. In this s tudy, we have examined the role of cytosine in more detail with the antigen omic ribozyme, Evidence that proton transfer in the rate-determining step i nvolved cytosine 76 (C76) was obtained from examining cleavage activity of the wild-type and imidazole buffer-rescued C76-deleted (C76 Delta) ribozyme s in D2O and H2O. In both reactions, a similar kinetic isotope effect and s hift in the apparent pKa indicate that the buffer is functionally substitut ing for the side chain in proton transfer, Proton inventory of the wild-typ e reaction supported a mechanism of a single proton transfer at the transit ion state. This proton transfer step was further characterized by exogenous base rescue of a C76 Delta mutant with cytosine and imidazole analogues. F or the imidazole analogues that rescued activity, the apparent pKa of the r escue reaction, measured under k(cat)/K-M conditions, correlated with the p Ka of the base. From these data a Bronsted coefficient (beta) of 0.51 was d etermined for the base-rescued reaction of C76 Delta This value is consiste nt with that expected for proton transfer in the transition state. Together , these data provide strong support for a mechanism where an RNA side chain participates directly in general acid or general base catalysis of the wil d-type ribozyme to facilitate RNA cleavage.