A further investigation and reappraisal of the thio effect in the cleavagereaction 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 re spective pro-Sp and pro-Rp oxygen atoms were replaced by sulfur, and subjec ted them to detailed kinetic analysis in the cleavage reaction catalyzed by a hammerhead ribozyme, In agreement with previous findings, in the presenc e of Mg2+ or Ca2+ ions the rate of ribozyme-catalyzed cleavage of S11SpS wa s as high as that of S11O, whereas the corresponding rate for S11RpS was ne arly four orders of magnitude lower than that for either S11O or S11SpS. Ho wever, the rate of the ribozyme-catalyzed reaction with each of the three s ubstrates was enhanced by Cd2+ 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 Cd2+ ion. However, our pre sent analysis demonstrates that (i) the added Cd2+ ion binds at the P9 site ; (ii) the bound Cd2+ ion at the P9 site replaces two Mg2+ or two Ca2+ ions , an observation that suggests a different mode of interaction with the add ed Cd2+ ion; and, most importantly and in contrast to the conclusion reache d by other investigators, (iii) the Cd2+ ion does not interact with the sul fur atom at the Rp position of the scissile phosphate either in the ground state or in the transition state.