Divalent Metal

Divalent metal ions, in addition to stabilizing overall structure, appear to have a more direct catalytic role in the cleavage reactions of the HDV ribozymes. Both HDV ribozymes are active in low (1-10 mM) concentrations of physiologically relevant divalent metal ions such as Mg2+ or Ca2+ (Wu et al. 1989; Perrotta and Been 1990; Rosenstein and Been 1990; Suh et al. 1993; Nakano et al. 2003). Altered metal ion specificity for cleavage of a 2'-5' linked phos-phodiester bond suggested a possible intimate role for the divalent metal ion in the cleavage reaction (Shih and Been 1999). High concentrations (1-2 M) of NaCl will support cleavage activity, but it is much slower than that measured in 2-10 mM Mg2+ (Nakano et al. 2000; Wadkins et al. 2001). The exchange-inert cation complex, Co(III) hexamine, which structurally mimics a hexahydrated Mg2+, does not support cleavage activity and inhibits the Mg2+-dependent reaction (Nakano et al. 2000; Ke et al. 2004). More recently, structures of precursor forms of a mutant ribozyme revealed a metal ion in the active site, and that metal ion is lost when the ribozyme cleaves (Ke et al. 2004). Together, these data would be consistent with the involvement of a divalent metal ion in catalysis.

Divalent metals tend to coordinate to the RNA through a water (outer-sphere coordination) but they also have the potential to coordinate directly to an oxygen or nitrogen in the RNA (inner-sphere coordination). An exhaustive kinetic and thermodynamic study of divalent-metal ion requirements of the genomic HDV ribozyme provided data that was evidence for both inner and outer-sphere binding at different metal sites (Nakano et al. 2003); a catalytic site that is strictly outer-sphere coordination but structural sites that also involve inner-sphere coordination. The absence of evidence for inner-sphere coordination of a catalytic metal ion was seen in other experiments as well. One approach to obtaining evidence for direct coordination of a metal ion to a scissile phosphate, nonbridging oxygen, is cleavage-rescue of thiophosphate-substituted RNA with a soft metal ion (e.g., Mn2+ or Cd2+). Although, in Mg2+, inhibition is seen with the pro-Rp thiophosphate, soft-metal ion rescue in the HDV ribozymes was not observed (Fauzi et al. 1997). Das and Piccirrili (2005) prepared a substrate oligonucleotide with a sulfur substituted for the 5'-bridging oxygen and found that a soft metal ion (Mn2+) did not stimulate the ribozyme-mediated cleavage even though it did stimulate the background cleavage of the same substrate. This result indicates that the metal ion does not directly coordinate to the phosphate 5'-bridging oxygen (the leaving group atom) in the ribozyme. Thus, so far at least, there is no evidence for inner-sphere coordination of a metal ion with catalytic activity.

A catalytic role that involves outer sphere coordination is feasible because a water molecule coordinated to a metal ion has a lower pKa than bulk water, thus raising the possibility that the hydrated metal ion may function as a general acid-base catalyst. Nakano et al. (2001, 2003) propose that proton transfer catalyzed by a hydrated Mg2+ contributes about 25-fold to the overall rate in a reaction containing 1 M NaCl. Two specific models for the hydrated metal ion acting as a general acid-base catalyst in the HDV ribozyme reaction have now been proposed. In one model it is acting at the 2' hydroxyl group (Nakano et al. 2000), and in the other it is acting at the 5' bridging oxygen (Ke et al. 2004). In the structure of the precursor, a hydrated metal ion is well positioned to coordinate through a water to the 5' oxygen leaving group and thus maybe a good candidate for a proton donor (Ke et al. 2004). On the other hand, in the earlier structure of the cleavage product, it appears that Cyt75 in the active site is well positioned for this same role (Ferre-D'Amare et al. 1998a; Nakano et al. 2000). Kinetic data alone is unavoidably ambiguous with regard to assigning the position at which an acid-base catalyst might act in a reaction such as the one being studied here because the two mechanisms would be kinetically equivalent. However, Das & Piccirrili (2005) have recently shown that cobalt hexamine was effective in inhibiting the cleavage reaction even when the 5' bridging oxygen was replaced by sulfur, a much better leaving group. This result suggests that the inhibition by cobalt hexamine, which is a poor proton donor/acceptor, is not the result of it replacing a critical hydrated Mg2+ that acts at the 5' leaving group as a proton donor. Indeed they find that Cyt76 likely fulfills that role in the antigenomic form of the ribozyme they used (described below).

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