An Active Site Nucleobase

Possible roles for a nucleobase in catalysis could range from the effective and ordinary (positioning reactive groups through hydrogen bond interactions) to the more exotic and elusive (general acid-base catalysts) to something in between (electrostatic shielding). Biochemical evidence and the crystal structures have led to the hypothesis that a cytosine located in the active site of the HDV ribozyme acts as a general acid-base catalyst (Tanner et al. 1994; Ferre-D'Amare et al. 1998a; Perrotta et al. 1999b; Nakano et al. 2000; Shih and Been 2001b; Ke et al. 2004). Early mutagenesis results suggested that Cyt75 in the genomic HDV ribozyme and Cyt76 at the equivalent position in the antigenomic ribozyme may have an important role in the cleavage reaction (Tanner et al. 1994; Perrotta and Been 1996). Lower, but measurable, activity could be detected with the cytosine to adenine substitution (Perrotta and Been 1996; Perrotta et al. 1999b; Nakano et al. 2000). However, it was the structure of the cleaved genomic ribozyme that provided the strongest support for cytosine playing a specific catalytic role (Ferre-D'Amare et al. 1998a). In the cleaved RNA, the cytosine is well positioned in the active site to act as a catalytic group, and both the O2 and N3 of Cyt75 are within hydrogen-bonding distance to the 5' hydroxyl group oxygen generated following the cleavage reaction. A specific model with the nucleobase of Cyt75 acting as a general acid-base catalyst and accepting a proton from the 2' hydroxyl group was proposed (Fig. 2b) (Ferre-D'Amare et al. 1998a). Shortly thereafter, a variation of that model was proposed (Nakano et al. 2000) with Cyt75 still acting as an general acid-base catalyst but donating a proton at the 5' bridging oxygen, the leaving group (Fig. 2c). The product structure would appear to be most consistent with Cyt75 acting at the 5' bridging oxygen leaving group but since the cleaved ribozyme lacked the 5' product, evidence for positioning of the 2' hydroxyl and phosphate groups was not available.

Thus, both models propose a role for the Cyt75 side chain that is analogous to the catalytic role of the imidazole group of histidine in RNase A. However, unlike RNase A which has two histidines in the active site that can alternatively function as proton acceptors and proton donors in the two steps catalyzed by RNase A, the active site of the HDV ribozyme may contain only the single catalytic nucleobase. If we continue with the RNase A comparison, a third possible catalytic role for Cyt75 would be equivalent to the lysine side chain stabilizing charge on the cleavage-site phosphoryl group. In the ribozyme this might be through either the exocyclic amino group protons or, in the ionized form of the nucleobase, an N3 proton. Answers to the following questions would help distinguish between possible roles for Cyt75/76. First, is there evidence that the cleavage rate is linked to the ionization state (protonation) of Cyt75/76. If so, is there evidence that Cyt75/76 is actually acting as a general-acid-base catalyst. And, if it is, at what position is the cytosine acting to accept or donate a proton? For the latter, the possibilities are again the 2' hydroxyl group, the 5' leaving group, or maybe a phosphate oxygen.

Results from several labs are consistent with the idea that the ionization state of Cyt75/76 is linked to cleavage activity. Differences in the pH-rate curves of the wild-type and Cyt75/76 to adenine mutants in both the antige-nomic and genomic ribozymes are consistent with that hypothesis (Perrotta et al. 1999b; Nakano et al. 2000). More direct evidence came from nucleotide analog interference mapping (NAIM) experiments (Oyelere and Strobel 2000) in which the effect of analogs with different ionization constants were examined for their effect on cleavage activity. Recently, Das and Piccirilli (2005) showed a clear shift in the apparent pKa of the cleavage reaction when 6-azacytosine replaced C76 in an antigenomic ribozyme. Those data all revealed a correlation of the ionization of a base at position 75/76 and self-cleavage activity.

The evidence that the cytosine is actually functioning as a general acid-base catalyst is indirect and requires correlating cleavage rates with the strength of the base or acid. Cleavage activity of antigenomic ribozyme variants in which Cyt76 was either changed to a U or deleted were partially rescued with the addition of free cytosine to the reaction (Perrotta et al. 1999b; Shih and Been 2001b). The rate of cleavage in the cytosine rescue reactions was both concentration and pH dependent. Base (buffer) rescue was also seen with imidazole and certain imidazole-like compounds with different pKa values (Shih and Been 2001b), and the shapes of the pH-rate curves for these rescue reactions were consistent with the idea that the cleavage rate reflected the ionization state of the base. More importantly, the reaction showed a dependence on the strength of the buffer which was consistent with general acid-base catalysis. A linear free-energy plot, the log of the second order rate constant for the base-rescued cleavage reaction versus pKa of the base, gave a line with a slope of ~0.5. This value is the Br0nsted coefficient (P) and, for this reaction, is consistent with an even distribution of charge between the proton donor and acceptor in the transition state; in other word the proton is 'in-flight' and moved about half-way in the transition state and thus consistent with a mechanism of general acid-base catalysis (Jencks 1969;Fersht 1985). Strictly speaking, the conclusion from this analysis only applies to the buffer-rescue reactions; the assumption used here, to argue for general acid-base catalysis by Cyt75(76), is that free cytosine and the imidazole compounds fulfill a similar functional role in the mutants as Cyt75(76) does in the wild-type ribozyme.

The base-rescue result, while providing strong support for a general acid-base mechanism, does not address the issue of where the general acid-base catalyst acts in the reaction. In this case, we cannot distinguish between the cytosine (or imidazole) acting to accept a proton from the 2' hydroxyl group (general base), or its conjugate acid donating a proton to the 5' oxygen leaving group (general acid). Ambiguity in assigning the site of action of a general acid-base catalyst from kinetic studies alone is unavoidable in this type of reaction (Jencks 1969).

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