Explanation by a putative triester-like mechanism for the thio effects and Mn2+ rescues in reactions catalyzed by a hammerhead ribozyme.

Divalent metal ion-dependent hammerhead ribozymes can cleave any RNA with a NUX triplet, wherein the N can be any residue and X can be C, U or A. In recent literature on the mechanism of action of hammerhead ribozymes, one important role of divalent metal ions is generally suggested to be an electrophilic catalyst by directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. This proposal was made on the basis of thio effects and the proposed electrophilic catalyst is very attractive as an explanation for the catalytic activity of metalloenzymes. Reexamination of thio effects with substrates having a GUA triplet at the cleavage site shows that, in agreement with the previous finding, the cleavage rate, in the presence of Mg2+ ions, is significantly reduced in the case of the phosphorothioate substrate (RpS), wherein the pro-Rp oxygen at the scissile phosphate is replaced by sulfur, while the cleavage rate is reduced to a much lesser extent for the other isomer (SpS), wherein the pro-Sp oxygen at the scissile phosphate is replaced by sulfur. However, more careful examination of the rescue ability of Mn2+ ions with these isomers demonstrates that more thiophilic Mn2+ ions rescue the reaction not only with the RpS isomer but also with the SpS isomer and, importantly, to a greater extent for the SpS isomer. These results argue against the previous conclusion that a metal ion is directly coordinating with the pro-Rp oxygen of the scissile phosphate to stabilize the transition state. In this paper we try to elucidate the possible origin of the thio effects and propose a 'triester-like' mechanism in reactions catalyzed by hammerhead ribozymes.