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 thio-substituted substrates S11 S pS and S11 R pS, in which the respective pro-S p and pro-R p 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 S11 S pS was as high as that of S11O, whereas the corresponding rate for S11 R pS was nearly four orders of magnitude lower than that for either S11O or S11 S pS. 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 R p 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 R p position of the scissile phosphate either in the ground state or in the transition state.