A new metal ion interaction in the Tetrahymena ribozyme reaction revealed by double sulfur substitution

The Tetrahymena ribozyme is a metalloenzyme that catalyzes cleavage of olig onucleotide substrates by phosphoryl transfer. Thiophilic metal ions such a s Mn2+, Zn2+ or Cd2+ rescue the >10(3)-fold inhibitory effect of sulfur sub stitution of the 3'-oxygen leaving group but do not effectively rescue the effect of sulfur substitution of the nonbridging pro-Sp phosphoryl oxygen. We now show that the latter effect can be fully rescued by Zn2+ or Cd2+ usi ng a phosphorodithioate substrate, in which both the 3'-oxygen and the pro- Sp oxygen are simultaneously substituted with sulfur. These results provide the first functional evidence that metallophosphotransferases can mediate catalysis via metal ion coordination to both the leaving group and a nonbri dging oxygen of the scissile phosphate.