A further investigation and reappraisal of the thio effect in the cleavage reaction catalyzed by a hammerhead ribozyme

We synthesized three types of 11mer substrate, namely the natural substrate S11O and the thiosubstituted substrates S11SpS and S11RpS, in which the respective pro-Sp and pro-Rp oxygen atoms were replaced by sulfur, and subjected them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme. In agreement with previous findings, in the presence of Mg(2+) or Ca(2+) ions the rate of ribozyme-catalyzed cleavage of S11SpS was as high as that of S11O, whereas the corresponding rate for S11RpS was nearly four orders of magnitude lower than that for either S11O or S11SpS. However, the rate of the ribozyme-catalyzed reaction with each of the three substrates was enhanced by Cd(2+) ions. Such results have generally been taken as evidence that supports the direct interaction of the sulfur atom at the Rp position of the cleavage site with the added Cd(2+) ion. However, our present analysis demonstrates that (i) the added Cd(2+) ion binds at the P9 site; (ii) the bound Cd(2+) ion at the P9 site replaces two Mg(2+) or two Ca(2+) ions, an observation that suggests a different mode of interaction with the added Cd(2+) ion; and, most importantly and in contrast to the conclusion reached by other investigators, (iii) the Cd(2+) ion does not interact with the sulfur atom at the Rp position of the scissile phosphate either in the ground state or in the transition state.