Site of Action of the Catalytic Cytosine

Although kinetic studies alone cannot identify the site at which the catalytic cytosine acts, structural studies and mechanistic studies with carefully chosen atom substitutions can. Structural data addressing this issue was discussed above. The recent mechanistic study by Das and Piccirrili (2005) specifically investigate the question of whether the active-site cytosine is acting at the 5' leaving group oxygen. To test that hypothesis, the 5' bridging oxygen was replaced with sulfur, which is a better leaving group, and the effect that substitution had on the reaction with and without the cytosine was examined. The logic behind this approach is that if the cytosine, acting as a general-acid catalyst, is donating a proton to the 5' bridging oxygen to make it a better leaving group in the cleavage reaction, then replacing the poor leaving group (oxygen) with a good leaving group (sulfur) will make the reaction less sensitive to the strength of the acid (Jencks 1969). The authors used an antigenomic form of the ribozyme with and without a C76U mutation and, in an impressive set of experiments, demonstrated that the 5' bridging sulfur 'rescues' cleavage activity in the C76U mutant background but had little effect in the wild-type background. Similar results were obtained with a 3-deazacytosine derivative at position 76 (C76c3C). The effect of 3-deazacytosine more precisely identifies the suspected N3 atom of cytosine as the site of ionization. Chemogenetic suppression (Das and Piccirilli, 2005) aptly describes this approach because the deleterious effect of the cytosine to uracil (or 3-deazacytosine) mutation was suppressed by the oxygen to sulfur substitution. This was a beautiful test of the hypothesis that Cyt76 was acting as a proton donor in the cleavage reaction and provides strong support for that specific role.

The interpretation of the most recent structural evidence (Ke et al. 2004) and the mechanistic data (Das and Piccirilli, 2005) thus provides two different models for the role of cytosine in the cleavage reaction. The structural data for the precursor form of the genomic ribozyme appears most consistent with the Cyt75 acting at the 2' hydroxyl group as a general base. The chemo-genetic suppression with the antigenomic ribozyme is most consistent with Cyt76 acting at the 5' oxygen leaving group as a general acid. Although two different forms of the ribozymes (genomic and antigenomic) were studied, the ribozymes are so similar this is very unlikely to be an issue. Both studies relied heavily on an equivalent cytosine to uracil mutation in the ribozyme, so the mechanisms would not differ as a result of the mutation. The possibility that the cytosine could act at both positions, perhaps transferring a proton from the 2' oxygen nucleophile to the 5' bridging oxygen leaving group, would seem unlikely for this particular reaction given the distance involved. So can we say where the cytosine acts? Given the difficulties in obtaining accurate high-resolution structures of precursor forms of the HDV ribozymes, it may not be possible to solve a crystal structure of a precursor ribozyme that is not subject to criticism at some level. Modifications have to be made to the sequence or conditions to prevent it from cleaving. One might likewise argue that replacing an oxygen with sulfur alters the mechanism of the reaction but the internal consistency of the effect of substitutions in the chemogenetic suppression data appear very strong. Nevertheless, debate on this detail of the mechanism is likely to continue.

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